key: cord-0769001-27r64von authors: Lassaunière, Ria; Polacek, Charlotta; Fonager, Jannik; Bennedbæk, Marc; Boding, Lasse; Rasmussen, Morten; Fomsgaard, Anders title: Neutralisation of the SARS-CoV-2 Delta variant sub-lineages AY.4.2 and B.1.617.2 with the mutation E484K by Comirnaty (BNT162b2 mRNA) vaccine-elicited sera, Denmark, 1 to 26 November 2021 date: 2021-12-09 journal: Euro Surveill DOI: 10.2807/1560-7917.es.2021.26.49.2101059 sha: 0a6edb54ec5edd8609185ac50c268b6577a3d1f6 doc_id: 769001 cord_uid: 27r64von Several factors may account for the recent increased spread of the SARS-CoV-2 Delta sub-lineage AY.4.2 in the United Kingdom, Romania, Poland, and Denmark. We evaluated the sensitivity of AY.4.2 to neutralisation by sera from 30 Comirnaty (BNT162b2 mRNA) vaccine recipients in Denmark in November 2021. AY.4.2 neutralisation was comparable to other circulating Delta lineages or sub-lineages. Conversely, the less prevalent B.1.617.2 with E484K showed a significant more than 4-fold reduction in neutralisation that warrants surveillance of strains with the acquired E484K mutation. Several factors may account for the recent increased spread of the SARS-CoV-2 Delta sub-lineage AY.4.2 in the United Kingdom, Romania, Poland, and Denmark. We evaluated the sensitivity of AY.4.2 to neutralisation by sera from 30 Comirnaty (BNT162b2 mRNA) vaccine recipients in Denmark in November 2021. AY.4.2 neutralisation was comparable to other circulating Delta lineages or sub-lineages. Conversely, the less prevalent B.1.617.2 with E484K showed a significant more than 4-fold reduction in neutralisation that warrants surveillance of strains with the acquired E484K mutation. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant of concern (VOC) (Phylogenetic Assignment of Named Global Outbreak Lineages (Pangolin) designation B.1.617) sub-lineage AY.4.2 recently accounted for an increased proportion of coronavirus disease (COVID-19) cases caused by the Delta variant in the United Kingdom (UK), increasing from 3.8% to 20.3% in the weeks of 19 September 2021 and 15 November 2021, respectively [1, 2] . The Delta sub-lineage AY.4.2 has spread globally, evidenced by over 48,000 sequences uploaded onto GISAID from 42 countries [3] and frequencies above 1% observed in Romania and Poland [4] as at 23 November 2021. A Delta strain bearing a known neutralisation-resistant mutation, E484K [5, 6] , has also established small clusters in the UK [7] and contributed to breakthrough infections in Italy [8] . Here, we investigate the sensitivity of the Delta sub-lineage AY.4.2 and Delta lineage B.1.617 with the E484K mutation (+ E484K) to neutralisation by SARS-CoV-2 Comirnaty (BNT162b2 mRNA, BioNTech-Pfizer, Mainz, Germany/New York, United States (US)) vaccine-induced anti-sera. SARS-CoV-2 virus neutralisation capacity was assessed using anti-sera from Danish residents vaccinated with Comirnaty, the predominant SARS-CoV-2 vaccine used in Denmark (85%). The cohort (n = 30), vaccinated with two doses between 18 January and 15 May 2021, was selected to represent a broad age range (20-91 years; median: 42 years; interquartile range (IQR): 35-63; 7/30 donors were men) and varied neutralisation reactivity against an early pandemic strain that is highly homologous to the vaccine strain (D614G; Table) [9] . The time between the second vaccination and sampling ranged from 30 to 100 days (median: 61 days; IQR: 51-64). All donors developed anti-SARS-CoV-2 spike antibodies after vaccination according to the Wantai Total Ab ELISA assay (Beijing Wantai Biological Pharmacy, Beijing, China) using the manufacturer's recommended cut-off for positivity [10, 11] . The samples were excess material from diagnostic testing conducted at Statens Serum Institut, Denmark. Samples were taken for diagnostic purposes unrelated to COVID-19; however, they are linked to the Danish Vaccination Registry, which enables surveillance of immune escape by vaccineinduced antibody responses. Virus neutralisation was tested against SARS-CoV-2 viruses isolated on Vero E6 cells (Table) . All virus stocks were sequenced to confirm the presence of lineage-specific mutations and the absence of cell culture-derived mutations. Two-fold serially diluted serum [12] . This SARS-CoV-2 microneutralisation assay has a comparable performance relative to other neutralisation assays used in European laboratories (Laboratory 4 in ref [13] ). The ELISA signal was unaffected by amino acid substitutions present in the nucleocapsid protein of some SARS-CoV-2 isolates. Exact 50% neutralisation titres were calculated using four-parameter logistic regression and titres below the lower limit of quantitation (1:10) were assigned a titre of 1:5. Titres were compared using the nonparametric Friedman test for paired measurements followed by Dunn's multiple comparison test. Adjusted p values are reported. The Delta sub-lineage AY.4.2 bears the same spike mutations as the Delta lineage AY.4 with the addition of Y145H and A222V in the N-terminal domain ( Figure 1 ). The A222V mutation occurred in SARS-CoV-2 variants that emerged in June 2020, but has been shown not to contribute to increased transmissibility or immune escape in those variants [14] . A functional consequence for Y145H remains to be established. Relative to the early pandemic strain (D614G), the AY.4.2 virus had a 2.3-fold reduction in median neutralisation titres (median titre: 199 vs 87; p < 0.001) (Figure 2 Virus neutralisation titres for other VOC, Alpha (Pango lineage designation B. [2] , an increase of 59 cases since an earlier report on 15 October 2021 [7] . Serum samples were each taken from a different individual (n = 30) and tested in a two-fold serial dilution ranging from 1:10-1:1,280. The dotted line represents limit of detection (< 1:10). *p < 0.05, **p < 0.01, ***p < 0.001. The study was conducted according to the Declaration of Helsinki. Biological samples were stored as part of the Statens Serum Institut's diagnostic testing and surveillance. Ethical approval was not required for this surveillance study. SARS-CoV-2 neutralising antibody titres against variants of concern Alpha, Beta, Gamma Moderna, Cambridge, Massachusetts, US) vaccines in the UK [7] . On the contrary, we show a significant neutralisation resistance for a Delta variant that acquired the E484K spike mutation. This amino acid substitution in the receptor-binding domain, occurring in other VOC such as Beta and Gamma, is associated with reduced sensitivity to monoclonal antibodies, convalescent antisera from early pandemic wave infections, and vaccine elicited anti-sera [6, 16, 17] . While Delta variants bearing the E484K mutation occur mostly sporadically [8] , it has now occurred in Delta sub-lineages with sustained clusters according to recent reports from the UK [7] . The presented data provide further support for continuous monitoring of E484K within emerging Delta sub-lineages, such as the Delta strain examined here. Additional studies of a larger sample size, which evaluate the antigenicity of VOC and circulating Delta variant strains against sera from children and adults vaccinated with different SARS-CoV-2 vaccines modalities (mRNA, recombinant adenovirus, subunit, inactivated) are warranted. These further include evaluations following booster vaccine doses after 4 to 6 months and following natural infection. In addition, similar studies are urgently needed for the recently identified Omicron VOC (Pango lineage designation B.1.1.529) and its sublineages [18] , which bear different spike mutations. SARS-CoV-2 variants of concern and variants under investigation in England SARS-CoV-2 variants of concern and variants under investigation in England COVID-19 Weekly Epidemiological Update -edition 63 Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization SARS-CoV-2 variants of concern and variants under investigation in England Breakthrough infections of E484K-harboring SARS-CoV-2 Delta variant Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine An evaluation of COVID-19 serological assays informs future diagnostics and exposure assessment Evaluation of nine commercial SARS-CoV-2 immunoassays Manual for the laboratory diagnosis and virological surveillance of influenza. Geneva: WHO SARS-CoV-2 neutralising antibody testing in Europe: towards harmonisation of neutralising antibody titres for better use of convalescent plasma and comparability of trial data Spread of a SARS-CoV-2 variant through Europe in the summer of 2020 E484K Mutation Report. Outbreak.info SARS-CoV-2 spike E484K mutation reduces antibody neutralisation Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7 Proposal to split B.1.1.529 to incorporate a newly characterised sibling lineage. GitHub Repository. GitHub We would like to thank Birgit Knudsen, Louise Borup, and Dennis Jelsbak Schmidt for technical assistance. We acknowledge the support from European Virus Archive GLOBAL project funded by the European Union's Horizon 2020 research and innovation program, grant agreement No 871029, for providing SARS-Related Coronavirus 2 strain hCoV-19/ Netherlands/NoordHolland_10159/2021 contributed by Chantal Reusken and Johannes Brug, RVIM, Bilthoven, NL. A preprint of the preliminary study is available on MedRxiv (doi: https://doi.org/10.1101/2021.11.08.21266075).Funding statement: No particular funding was obtained for this work, which was a part of the Danish national health response to the COVID-19 pandemic. None declared. This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made. Any supplementary material referenced in the article can be found in the online version.