key: cord-0781794-64vi0u0s authors: Lassauniere, R.; Polacek, C.; Fonager, J.; Bennedbaek, M.; Boding, L.; Rasmussen, M.; Fomsgaard, A. title: Neutralisation of SARS-CoV-2 Delta sub-lineage AY.4.2 and B.1.617.2+E484K by BNT162b2 mRNA vaccine-elicited sera date: 2021-11-09 journal: nan DOI: 10.1101/2021.11.08.21266075 sha: 53401e0acd2832a30c308d038e056b158108987b doc_id: 781794 cord_uid: 64vi0u0s A sublineage of the Delta variant, AY.4.2, recently accounted for an increase proportion of Delta cases in United Kingdom (UK), Romania, Poland, and Denmark. Several factors may account for the increased spread of AY.4.2. Here, we evaluated the sensitivity of AY.4.2 to neutralisation by sera from Pfizer/BioNTech (BNT162b2) vaccinees. AY.4.2 was not more resistant to neutralisation relative to other circulating Delta lineages or sublineages and showed only a modest 2.3-fold reduction in neutralisation relative to the vaccine strain. In contrast, the more rare B.1.617.2+E484K variant showed a 4.2-fold reduction in neutralisation that warrants surveillance of strains with the acquired E484K mutation. In recent weeks, the SARS-CoV-2 Delta sublineage AY.4.2 has accounted for an increased proportion of Delta cases in the United Kingdom (UK), rising from 3.8% to 11.3% in the weeks beginning with the 19 th of September 2021 and the 18 th of October 2021, respectively [1, 2] . Multiple other countries reported its presence with over 26 000 AY.4.2 sequences from 42 countries uploaded on GISAID to date [3] , and frequencies above 1% observed in Romania and Poland [4] . On the 20 th October 2021, the UK Health Security Agency designated AY.4.2 a Variant Under Investigation (VUI-21OCT-01) as it appears to have a higher growth rate (19%) in the population compared to other Delta lineages and sublineages [1] . AY.4.2 bears the defining spike mutations of AY.4 with the addition of Y145H and A222V. Neutralisation data for AY.4.2 is currently lacking and warrants rapid evaluation to establish if neutralisation resistance may account for the increased spread of this sublineage. We further evaluate neutralisation of a Delta strain bearing the known neutralisation resistant mutation, E484K [5, 6] . The latter amino substitution has sporadically occurred in Delta strains, but recently established small clusters in the UK [2] and contributed to breakthrough infections in Italy [7] . Here we investigate the sensitivity of AY.4.2 and the Delta lineage B.1.617+E484K to neutralisation by SARS-CoV-2 vaccineinduced anti-sera. We assessed SARS-CoV-2 Delta sublineage AY.4.2 virus neutralisation using serum samples from BNT162b2 (Pfizer/BioNTech) mRNA vaccinated individuals (N = 14) between the ages of 26 and 72 years (median: 42 years); 35.7% were male [8] . The time between the second vaccination and sampling ranged from 49 to 68 days (median: 63 days). All donors developed anti-SARS-CoV-2 spike antibodies after vaccination as determined by the Wantai Total Ab ELISA assay [9, 10] . The samples were excess material from diagnostic testing conducted at Statens Serum Institut, Denmark. Samples were not taken in relation to COVID-19, but are linked to the Danish Vaccination Registry that enables surveillance of immune escape by vaccine-induced antibody responses. Virus neutralisation was tested against SARS-CoV-2 clinical isolates cultured on Vero E6 cells (Table 1) . All virus stocks were sequenced to confirm the presence of lineage-specific mutations and the absence of cell culture-derived mutations. Inhibition of virus infection is measured in an anti-SARS-CoV-2 nucleocapsid protein ELISA. The quantitative optical density values from the two-fold serial dilution of serum (range: 1:10 to 1:1280) enables calculation of exact 50% neutralisation titers using fourparameter logistic regression. The assay has a comparable performance relative to other neutralisation assays used in different European laboratories (laboratory 4 in ref [11] ). Titers were compared using the Friedman test for paired measurements followed by Dunn's multiple comparison test. Adjusted P-values are reported. We first assessed AY.4.2 virus neutralisation by vaccine sera relative to other Delta sublineages bearing lineage and sublineage-defining spike protein amino acid changes as well as an early pandemic strain bearing a single spike amino acid change (D614G) ( Figure 1 ). All tests were performed concurrently to ensure minimal inter-assay variation. Relative to the early pandemic strain (D614G), the AY.4.2 virus had a 2.3-fold reduction in median neutralisation titers (median titer: 194 vs. 86; P = 0.003) ( Figure 2 Virus neutralisation titers for other VOC were available for 11 of the 14 serum samples (Figure 3 ). Relative to the early pandemic strain (D614G), the reduction in the AY.4.2 sublineage-associated virus neutralisation (2.3-fold) was not as pronounced as observed for the Beta variant (4.9-fold). In contrast, the Delta lineage B.1.617.2 with the E484K neutralisation resistant mutation had a comparable reduction in neutralisation titers observed for the Beta variant (4.2-fold). AY.4.2. and B.1.617.2 In Denmark, the first AY4.2 case was observed on the 4 th August 2021. In September 2021, AY4.2 appeared in more than 2% of sequenced samples but declined to below 1%. However, during the last week of October 2021, AY4.2 increased from 0.5% to 2%. The E484K mutation are only found sporadic in several different lineages other than the Beta and Gamma. To date, 34 cases of Delta strains with the E484K mutation has been identified in Denmark between the 29 th of August 2021 and the 28 th of October 2021. In addition to the spike mutations present in the prevalent AY.4 sublineage, AY.4.2 bears the Y145H and A222V amino acid substitutions. Both occur in the N-terminal domain of the spike protein. A222V has been observed in earlier emerging SARS-CoV-2 variants, but is not considered to contribute to increased transmissibility or immune escape in those variants [12] . A functional consequence for Y145H remains to be established. Using a live virus neutralisation assay, we demonstrate that the Delta sublineage AY.4.2 virus has a modest reduction of 2.3-fold relative to an early pandemic strain, which is highly homologous to the current vaccine strains. AY.4.2 therefore remains sensitive to vaccine-induced virus neutralisation. Moreover, neutralisation titers for AY.4.2 were not significantly different from the parental B.1.617.2 or AY.4 lineage. It is thus unlikely that neutralisation resistance is a determinant of the increased spread observed for AY.4.2 relative to other Delta lineages in European countries. These findings are in agreement with a similar vaccine effectiveness observed for AY4.2 compared to non-AY4.2 Delta cases, both symptomatic and asymptomatic, for the Astra Zeneca, Pfizer/BioNTech and Moderna vaccines in the UK [2] . 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, occur in other VOC that include Beta and Gamma with reduced sensitivity to monoclonal antibodies and vaccine elicited anti-sera [6, 13, 14] . While Delta variants bearing the latter mutation occur mostly sporadically [7] , it has occurred in Delta sublineages with sustained clusters recently reported in the UK [2] . The presented data provide further support for continuous monitoring of E484K within emerging Delta sublineages such as the Delta strain examined here. Biological samples were stored as part of the Statens Serum Institut's diagnostic testing and surveillance. No ethical approval was required for this surveillance study. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 9, 2021. ; is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 9, 2021. ; is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 9, 2021. ; is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 9, 2021. ; https://doi.org/10.1101/2021.11.08.21266075 doi: medRxiv preprint SARS-CoV-2 Variants of Concern and Variants under Investigation in England-Technical Briefing 26 SARS-CoV-2 Variants of Concern and Variants under Investigation in England-Technical Briefing 27 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 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 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 SARS-CoV-2 spike E484K mutation reduces antibody neutralisation. The Lancet Microbe 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 programme, 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.