Repeated Omicron exposures override ancestral SARS-CoV-2 immune imprinting

The continuing emergence of SARS-CoV-2 variants highlights the need to update COVID-19 vaccine compositions. However, immune imprinting induced by vaccination based on the ancestral (hereafter referred to as WT) strain would compromise the antibody response to Omicron-based boosters1–5. Vaccination strategies to counter immune imprinting are critically needed. Here we investigated the degree and dynamics of immune imprinting in mouse models and human cohorts, especially focusing on the role of repeated Omicron stimulation. In mice, the efficacy of single Omicron boosting is heavily limited when using variants that are antigenically distinct from WT—such as the XBB variant—and this concerning situation could be mitigated by a second Omicron booster. Similarly, in humans, repeated Omicron infections could alleviate WT vaccination-induced immune imprinting and generate broad neutralization responses in both plasma and nasal mucosa. Notably, deep mutational scanning-based epitope characterization of 781 receptor-binding domain (RBD)-targeting monoclonal antibodies isolated from repeated Omicron infection revealed that double Omicron exposure could induce a large proportion of matured Omicron-specific antibodies that have distinct RBD epitopes to WT-induced antibodies. Consequently, immune imprinting was largely mitigated, and the bias towards non-neutralizing epitopes observed in single Omicron exposures was restored. On the basis of the deep mutational scanning profiles, we identified evolution hotspots of XBB.1.5 RBD and demonstrated that these mutations could further boost the immune-evasion capability of XBB.1.5 while maintaining high ACE2-binding affinity. Our findings suggest that the WT component should be abandoned when updating COVID-19 vaccines, and individuals without prior Omicron exposure should receive two updated vaccine boosters.


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Policy information about availability of computer code Data collection Pseudovirus neutralization and ELISA data were collected by Multiskan™ FC Microplate Photometer.SPR data was collected by BIAcore 8K Evaluation Software (v4.0.8.20368;GE Healthcare).FACS data was collected by Summit 6.0 (Beckman Coulter).
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Sample size
A total of 1816 antibodies were characterized in the manuscript.We analyzed all antibodies in hand and the sample size of antibodies in this study was sufficient to reach statistical significance by two-tailed binomial test for the differences in epitope distribution.We collected plasma samples from 50 convalescent individuals with BA.1 breakthrough infection, 22 long-term convalescent individuals with BA.1 breakthrough infection, 39 with BA.2 breakthrough infection, 36 with BA.5 breakthrough infection and 30 with BF.7 breakthrough infection, all of whom received three doses of CoronaVac before infection.Further, we investigated 26 individuals who had post-vaccination BA.1 breakthrough infection followed by BA.5/BF.7 reinfection, 19 with post-vaccination BA.2 breakthrough infection followed by BA.5/BF.7 reinfection, and 12 individuals with BA.1/BA.2infection followed by BA.5/BF.7 reinfection, who had no history of vaccination.We analyzed all plasma samples collected and the sample size of plasma could reach statistical significance of NT50 values from neutralization assays by two-tailed Wilcoxon signed-rank test.
No sample size calculation was performed.
Data exclusions 466 antibodies were excluded from the study because of insufficient antibody or failed deep mutational scanning experiments, which is defined as no mutations scored two times of the median score.

Replication
Experimental assays were performed in at least two independent experiments according to or exceeding standards in the field.Specifically, we performed mutation screening using two independently constructed mutant libraries.We conducted all neutralization assays and ELISA in at least two independent experiments.All replicates for neutralization and ELISA are successful.
Randomization Randomization was not required since we were applying a uniform set of measurements across the panel of monoclonal antibodies and plasma.As this is an observational study, randomization is not relevant.

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Blinding was not required since we were applying a uniform set of measurements across the panel of monoclonal antibodies and plasma.As this is an observational study, investigators were not blinded.
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