Neutralization, effector function and immune imprinting of Omicron variants

Currently circulating SARS-CoV-2 variants have acquired convergent mutations at hot spots in the receptor-binding domain1 (RBD) of the spike protein. The effects of these mutations on viral infection and transmission and the efficacy of vaccines and therapies remains poorly understood. Here we demonstrate that recently emerged BQ.1.1 and XBB.1.5 variants bind host ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1, XBB.1 and BN.1 RBDs bound to the fragment antigen-binding region of the S309 antibody (the parent antibody for sotrovimab) and human ACE2 explain the preservation of antibody binding through conformational selection, altered ACE2 recognition and immune evasion. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1 and hamsters challenged with XBB.1.5. Vaccine-elicited human plasma antibodies cross-react with and trigger effector functions against current Omicron variants, despite a reduced neutralizing activity, suggesting a mechanism of protection against disease, exemplified by S309. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring the role of persistent immune imprinting.

Findings do not apply to only one sex or gender.Sex and/or gender were not considered in study design.Sex and/or gender was determined on self-reporting.Individual sex and/or gender information is shown as coded in Table S5 and S6 for all the participants analyzed.Sex and/or gender-based analyses were not performed as they are not relevant for this study.

Population characteristics
Individuals who received 3 or 4 doses of the Wuhan-Hu-1 monovalent mRNA vaccine or the Wuhan-Hu-1/BA.5 or Wuhan-Hu-1/BA.1 bivalent mRNA vaccines.Demographic data is provided in Tables S6 and S7.

Recruitment
Enrolled in the HAARVI study or under study protocols approved by the local institutional review boards.The participation to the study is voluntary so there might be a self-selection bias that cannot be eliminated.However, since this bias is present in all the cohorts analyzed, it is not expected to impact the results.

Ethics oversight
of Washington Human Subjects Division Institutional Review Board (STUDY00000959) and Canton Ticino and Canton Aargau Ethics Committees, Switzerland.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Life sciences study design
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Sample size
For in vivo studies the sample size was chosen based on prior experience with these animal models and previous publications with human cohorts.
Data exclusions No data were excluded from the analysis.

Replication
Experimental assays were performed at least in two independent replicates.Each replicates was performed with 2 or more technical replicates.All attempts at replication were successful Randomization Randomization and blinding were not possible due to pre-defined housing conditions (separated isolators between infected and non-infected animals).

Blinding
Ex vivo analyses were blinded (coded samples).

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Flow Cytometry Plots
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A numerical value for number of cells or percentage (with statistics) is provided.
Spike Expression: Transiently transfected BHK-21-GFP1-10 cells were collected by centrifugation at 1,000 x g for 5 min.The cells were washed once with PBS and fixed with 2% paraformaldehyde.The cells were washed twice with flow staining buffer (1% BSA, 1 mM EDTA, 0.1% NaN3 in PBS) and labeled with 250 μg/mL of S2L20, an NTD-directed antibody that recognizes all currently and previously circulating SARS-CoV-2 variants, for 45 minutes.The cells were washed three times with flow staining buffer and labeled with a PE-conjugated anti-Human IgG Fc antibody (Thermo Fisher) for 30 mins.The cells were washed an additional three times and resuspended in flow staining buffer.

Cell population abundance
The frequency and counts of memory B cells are provided in Extended Data Fig9.
Gating strategy PBMC Analysis: Lymphocytes were selected based on SSC-A vs FSC-A.Single cells were selected based on FSC-H vs FSC-A.Live cells were then selected based on FSC-A vs Zombie-Aqua with live cells being Aqua negative/low.B cells were then selected based on CD20-PECy7 vs CD3/8/14/16-Alexa eFluor780 with the positive population being PECy7 high and CD3/8/14/16 negative.Memory B cells were selected based on CD38-BV785 vs IgD/M-Alexa647 with memory B cells being BV785 negative/low and Alexa647 negative/low.Memory B cells binding streptavidin-biotin were selected based on FSC-A vs Streptavidin-biotin-BV711 with cells not binding streptavidin-biotin (BV711 negative) being selected.RBD-binding memory B cells were identified based on Omicron RBD pool-BV421 vs Wuhan-Hu-1 RBD-Dylight 488 with BV421 and/or Dylight 488positive cells being selected.Final quadrant gates were set on the RBD-positive memory B cells to determine specificity based on Omicron RBD pool-BV421 vs Wuhan-Hu-1 RBD-Dylight 488 or BQ.1.1RBD-Alexa568 vs Omicron RBD pool-BV421.

Materials
samples No field collected samples were used in the study Ethics oversight Animal studies were carried out in accordance with the recommendations in the Guide for the are and Use of Laboratory Animals of the National Institutes of Health.The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381-01) or the Animal Experimentation Ethics Committee (CETEA 89) of the Institut Pasteur