Reverse mutational scanning of spike BA.2.86 identifies the epitopes contributing to immune escape from polyclonal sera

The recently detected Omicron BA.2.86 lineage contains more than 30 amino acid mutations relative to BA.2. Here, we identify the epitopes driving immune escape of BA.2.86 and its derivative JN.1 (BA.2.86 + S455L) lineage. We investigated the cross-reactive humoral immunity within a cohort of health care workers against Omicron BA.2.86 and JN.1 by employing pseudo-viral mutants. We demonstrate that BA.2.86 and especially JN.1 evaded neutralization by serum antibodies of fully vaccinated individuals. To discern the contribution of individual epitope mutations to immune escape, we constructed a library of 33 BA.2.86 mutants, each of which harbored a single revertant mutation going back to BA.2. This library was used in a reverse mutational scanning approach to define serum neutralization titers against each epitope separately. The mutations within the receptor binding domain (RBD) at position K356T and to a lesser extent the mutations N460K, V483Δ, A484K, and F486P enhanced the immune escape. More surprisingly, the mutation 16insMPLF within the spike N-terminal domain (NTD) and the mutation P621S in S1/S2 significantly contributed to antibody escape of BA.2.86. Upon XBB.1.5 booster vaccination, neutralization titers against JN.1 and BA.2.86 improved relative to all ancestral strains, and the residual immune escape was driven by mutations at positions 16insMPLF, Δ144Y, E544K, P621S, and A484K. ### Competing Interest Statement L.C-S. served as an advisor to Sanofi unrelated to this work. G.M.N.B. served as advisor for Moderna unrelated to this work, A.D-J served as an advisor for Pfizer unrelated to this work, S.P. and M.H. conducted contract research (testing of vaccinee sera for neutralizing activity against SARS-CoV-2) for Valneva unrelated to this work. S.P. served as advisor for BioNTech, unrelated to this work. H.J. served as advisor on COVID neutralization assays for WHO and CEPI, unrelated to this work. The other authors declare no competing interests. ### Funding Statement This research was funded by the Helmholtz Association through Helmholtz Campaign COVIPA (KA1-Co-02) to L.C-S. and EU-Partnering grant MCMVaccine (PIE-008) to L.C-S. and S.P. Funding to L.C-S and S.P. was also provided by the Ministry of Science and Culture of Lower Saxony, through the COFONI Network, flex fund projects 6FF22 and 10FF22. G.M.N.B. and A.D.-J. acknowledge funding from Ministry of Science and Culture of Lower Saxony (14-76103-184, COFONI Network, project 4LZF23), G.M.N.B. acknowledges funding by the European Regional Development Fund (ZW7-85151373), and A.D.-J. acknowledge funding by European Social Fund (ZAM5-87006761). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The collection of all plasma samples was approved by the research ethics committee of the Institutional Review Board of Hannover Medical School (8973 BO K 2020). All donors provided written consent for the blood donation and use for research purposes. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors