Omicron BA.1 breakthrough infection drives cross-variant neutralization and memory B cell formation against conserved epitopes

Omicron is the evolutionarily most distinct SARS-CoV-2 variant of concern (VOC) to date. We report that Omicron BA.1 breakthrough infection in BNT162b2-vaccinated individuals resulted in strong neutralizing activity against Omicron BA.1, BA.2 and previous SARS-CoV-2 VOCs, but not against the Omicron sublineages BA.4 and BA.5. BA.1 breakthrough infection induced a robust recall response, primarily expanding BMEM cells against epitopes shared broadly amongst variants, rather than inducing BA.1-specific B cells. The vaccination-imprinted BMEM cell pool had sufficient plasticity to be remodeled by heterologous SARS-CoV-2 spike glycoprotein exposure. While selective amplification of BMEM cells recognizing shared epitopes allows for effective neutralization of most variants that evade previously established immunity, susceptibility to escape by variants that acquire alterations at hitherto conserved sites may be heightened.


INTRODUCTION
Containment of the coronavirus disease 2019 (COVID-19) pandemic requires the generation of durable and sufficiently broad immunity to provide protection against current and future variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The titer of neutralizing antibodies to SARS-CoV-2 and the binding of antibodies to the spike (S) glycoprotein and its receptor binding domain (RBD) are considered correlates of protection against infection (1,2). Currently available vaccines are based on the S glycoprotein of the ancestral Wuhan-Hu-1 strain and induce antibodies with a neutralizing capacity that exceeds the breadth elicited by infection with the Wuhan strain or with variants of concern (VOCs) (3). However, protective titers wane over time (4)(5)(6)(7), and routine booster vaccinations are thought to be needed to trigger recall immunity and maintain efficacy against new VOCs (8)(9)(10)(11).
Long-lived memory B (B MEM ) cells are the basis for the recall response upon antigen reencounter either by infection or booster vaccination. They play an important role in the maintenance and evolution of the antiviral antibody response against variants, because low-affinity selection mechanisms during the germinal center reaction and continued hypermutation of B MEM cells over several months after antigen exposure expand the breadth of viral variant recognition (12,13).
To date, more than 1 billion people worldwide have been vaccinated with the mRNA-based COVID-19 vaccine BNT162b2 and have received the primary two-dose series or further boosters (14). Thus, BNT162b2 vaccination is contributing substantially to the pattern of population immunity in many regions of the world.
How vaccine-mediated protective immunity will evolve over time and will be modified by iterations of exposure to COVID-19 vaccines and to infections with increasingly divergent viral variants remains poorly understood and is of particular relevance with the emergence of antigenically distinct VOCs. Omicron is the evolutionary most distant reported VOC to date, with a hitherto unprecedented number of amino acid alterations in its S glycoprotein, including at least 15 amino acid changes in the RBD and extensive changes in the N-terminal domain (NTD) (15). These alterations are predicted to affect most neutralizing antibody epitopes (16)(17)(18)(19)(20). In addition, Omicron is highly transmissible, has outcompeted Delta within weeks to become the dominant circulating VOC, and has given rise to multiple sublineages, starting with BA.1 and BA.2, that are spreading rapidly across the globe (21,22). New Omicron sublineages that harbor further alterations in the S glycoprotein continue to arise, with BA.4 and BA.5 deemed VOCs by the European Centre for Disease Prevention and Control on 12 May 2022 (23).
To characterize the effect of Omicron breakthrough infection on the magnitude and breadth of serum neutralizing activity and B MEM cells, we studied blood samples from individuals who were doubleor triple-vaccinated with BNT162b2, including cohorts who experienced breakthrough infection between November 2021 and mid-January 2022, a period when the BA.1 lineage was dominant in Germany (24). Because an understanding of the antigen-specific B cell memory pool is a critical determinant of an individual's ability to respond to newly emerging variants, our data will help to guide further vaccine development.
samples from vaccinated individuals with subsequent SARS-CoV-2 Omicron breakthrough infection experienced in a period of Omicron sublineage BA.1 dominance, and we therefore refer to "BA.1 breakthrough infection" herein. Samples were selected to investigate biomarkers in four independent groups, namely, individuals who were (i) double-or (ii) triple-vaccinated with BNT162b2 without a prior or breakthrough infection at the time of sample collection (BNT162b2 2 and BNT162b2 3 ) and individuals who were (iii) double-or (iv) triplevaccinated with BNT162b2 and who experienced breakthrough infection with the SARS-CoV-2 Omicron variant after a median of about 5 months or 4 weeks, respectively (BNT162b2 2 + Omi and BNT162b2 3 + Omi). Median ages of the cohorts were similar (32 to 39 years), except for the BNT162b2 2 cohort, who had a mildly increased median age of 52, albeit with only two individuals >65 years of age. Immune sera were used to characterize Omicron infection-associated changes to the magnitude and the breadth of serum neutralizing activity. Peripheral blood mononuclear cells (PBMCs) were used to characterize the VOC specificity of peripheral B MEM cells recognizing the respective full-length SARS-CoV-2 S glycoprotein or its RBD ( Fig. 1 and tables S1 to S3).

Omicron BA.1 breakthrough infection after BNT162b2 vaccination induces broad neutralization against Omicron
BA.1, BA.2, and other VOCs but not against BA.4 and BA. 5 To evaluate the neutralizing activity of immune sera, we used two orthogonal test systems: a well-characterized pseudovirus neutralization test (25,26) to investigate the breadth of inhibition of virus entry in a propagation-deficient setup and a live SARS-CoV-2 neutralization test designed to evaluate neutralization during multicycle replication of authentic virus with the antibodies maintained throughout the entire test period. For the former, we applied pseudoviruses bearing the S glycoproteins of SARS-CoV-2 Wuhan, Alpha, Beta, Delta, Omicron BA.1, and BA.2 and of the recently emerged Omicron sublineages BA.4 and BA.5 to assess neutralization breadth. Because BA.4 and BA.5 share an identical S glycoprotein sequence, including key alterations L452R and F486V, we herein refer to them as BA.4/5. In addition, we assayed SARS-CoV (herein referred to as SARS-CoV-1) to detect potential pan-sarbecovirus neutralizing activity (27).
Omicron BA.1 breakthrough infection had a marked effect on the magnitude and breadth of the neutralizing antibody responses of both double-and triple-vaccinated individuals, with slightly higher pVN 50 GMTs observed in the triple-vaccinated individuals ( Fig. 2A, fig. S1B, and  We observed a similar pattern when studying the neutralization of these variants with BA.1 convalescent and control sera from triplevaccinated individuals. BA.1 convalescent sera exhibited high pVN 50 GMTs against the previous SARS-CoV-2 VOCs, including Beta (1182), Omicron BA.1 (1029), and BA.2 (836) that were close to the Fig. 1. Cohorts, sampling, and experimental setup. Blood samples were drawn from four cohorts: Omicron-naïve individuals double-or triple-vaccinated with BNT162b2 (light and dark green) and individuals double-or triple-vaccinated with BNT162b2 who subsequently had a breakthrough infection with Omicron (light and dark purple) at the time of BA.1 dominance. PBMCs (red) and sera (yellow) were isolated in the Omicron-naïve cohorts at the time points indicated after their most recent vaccination; for convalescent cohorts, the time from their most recent vaccination to Omicron infection and infection to PBMC and serum isolation are indicated in purple text. All values are specified as median (range). The age/gender composition of cohorts is further detailed in tables S4 and S10. Serum neutralizing capacity was assessed using pseudovirus and live virus neutralization tests. SARS-CoV-2 S glycoprotein-specific B MEM cells were assessed via a flow cytometry-based B cell phenotyping assay using bulk PBMCs. N/A, not applicable. The schematic was created with BioRender.com.  In all cohorts, neutralizing titers against BA.4/5 were closer to the low level observed against the phylogenetically more distant SARS-CoV-1 than to that seen against Wuhan ( Fig. 2A and tables S4 to S6). Looking at the ratios of SARS-CoV-2 VOC and SARS-CoV-1 pVN 50 GMTs normalized against Wuhan, it is remarkable that breakthrough infection with Omicron BA.1 does not lead to more efficient crossneutralization of Omicron BA.4/5 in double-and triple-vaccinated individuals as compared with triple-vaccinated Omicron-naïve individuals (Fig. 2B).

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Authentic live SARS-CoV-2 virus neutralization assays conducted with Wuhan, Beta, Delta, and Omicron BA.

B MEM cells of BNT162b2 double-and triple-vaccinated individuals broadly recognize VOCs and are further boosted by Omicron BA.1 breakthrough infection
Next, we investigated the phenotype and quantity of SARS-CoV-2 S glycoprotein-specific B cells in these individuals. To this aim, we used flow cytometry-based B cell phenotyping assays for differential detection of variant-specific S glycoprotein-binding B cells in bulk PBMCs. We found that all S glycoprotein-and RBD-specific B cells in the peripheral blood were of a B MEM phenotype (B MEM ; CD20 high CD38 int/neg , fig. S3A). Antigen-specific plasmablasts or naïve B cells were not detected. The assays allowed us to identify B MEM  As expected, the overall frequency of antigen-specific B MEM cells varied across the different groups. Consistent with prior reports (30), the frequency of B MEM cells in Omicron-naïve double-vaccinated individuals was low at an early time point after vaccination and increased over time: At 5 months as compared with 3 weeks after the second BNT162b2 dose, S glycoprotein-specific B MEM cells almost quadrupled and RBD-specific ones tripled across all VOCs, thereby reaching quantities similar to those observed in Omicron-naïve triple-vaccinated individuals (Fig. 3, B and C; fig. S4, A to C; and table S12).
BNT162b2 double-or triple-vaccinated individuals with a SARS-CoV-2 Omicron BA.1 breakthrough infection exhibited a strongly increased frequency of S glycoprotein-specific B MEM cells, which was higher than those of Omicron-naïve triple-vaccinated individuals (Fig. 3 We then compared the ratios of RBD-to S glycoprotein-binding B MEM cells within the different groups and found that they are biased toward S glycoprotein recognition for the Omicron BA.1 VOC, particularly in the Omicron-naïve groups (Fig. 3F). In the Omicron BA.1 convalescent groups, this ratio was higher, indicating that an Omicron BA.1 breakthrough infection improved Omicron BA.1 RBD recognition.

Omicron BA.1 breakthrough infection after BNT162b2 vaccination boosts B MEM cells against epitopes broadly conserved across S glycoproteins of Wuhan and other VOCs
Our findings imply that Omicron BA.1 infection in vaccinated individuals not only boosts neutralizing activity and B MEM cells against Omicron BA.1 but also broadly augments immunity against various VOCs. To investigate the specificity of antibody responses at a cellular level, we performed multiparameter analyses of B MEM cells stained with fluorescently labeled variant-specific S or RBD proteins. By applying a combinatorial gating strategy, we sought to distinguish between B MEM cell subsets that could identify epitopes specific to a single variant only (either Wuhan, Alpha, Delta, or Omicron BA.1) versus those that could identify epitopes shared by any given combination of these variants ( Fig. 4A and fig. S3).
In the first analysis, we evaluated B MEM cell recognition of Wuhan and Omicron BA.1 S and RBD proteins (Fig. 4, B to D). Staining with full-length S glycoproteins showed that the largest proportion of B MEM cells from Omicron-naïve double-vaccinated individuals and even more predominantly from triple-vaccinated individuals were directed against epitopes shared by both Wuhan and Omicron BA.1 SARS-CoV-2 variants. Consistent with the fact that vaccination with BNT162b2 can elicit immune responses against Wuhan epitopes that do not recognize the corresponding altered epitopes in the Omicron BA.1 S glycoprotein (Fig. 4, B and C, and fig. S5A), we found in most individuals a smaller but clearly detectable proportion of B MEM cells that recognized only Wuhan S glycoprotein or RBD. Consistent with the lack of exposure, almost no B MEM cells binding exclusively to Omicron BA.1 S or RBD protein were detected in these Omicron-naïve individuals.
In Omicron BA.1 convalescent individuals, frequencies of B MEM cells recognizing S glycoprotein epitopes shared between Wuhan and Omicron BA.1 were considerably higher than in the Omicron-naïve ones (Fig. 4, B and C). This was particularly pronounced for double-vaccinated individuals. In most of these individuals, we also found a small proportion of exclusively Wuhan S glycoproteinspecific B MEM cells and a moderately lower frequency of exclusively Omicron BA.1 variant S glycoprotein-specific B MEM cells ( fig. S5A).
A slightly different pattern was observed by B cell staining with labeled RBD proteins (Fig. 4, B and D, and fig. S5B). Again, Omicron BA.1 breakthrough infection of double-/triple-vaccinated individuals was found to primarily boost B MEM cells reactive against conserved epitopes. A moderate boost of Wuhan-specific reactivities was observed; however, we detected only small populations of B MEM cells specific to the Omicron BA.1 RBD in the tested individuals ( Fig. 4D and fig. S5B).
Next, we used the combinatorial gating approach to identify the subsets of S glycoprotein-or RBD-binding B MEM cells that either bind exclusively to Wuhan or Omicron BA.1 or to common epitopes conserved broadly throughout all four variants, Wuhan, Alpha, Delta, and Omicron BA.1 (Fig. 4E). Across all four cohorts, we found that the frequency of B MEM cells recognizing S glycoproteinconserved epitopes accounted for the largest fraction of the pool of To further dissect the nuances of this response, we characterized the B MEM subsets directed against the RBD in both double-and triple-vaccinated Omicron BA.1 convalescent individuals. We used the combinatorial Boolean gating approach to discern B MEM cells with distinct binding patterns in the spectrum of strictly variant-specific and common epitopes shared by several variants. Multiple-sequence alignment revealed that the Omicron BA.1 RBD diverges from the RBD sequence regions conserved in Wuhan, Alpha, and Delta by 13 single-amino acid alterations (fig. S6). The most prominent B MEM cell population that we detected in BA.1 convalescent individuals recognized Wuhan, Alpha, and the Delta RBDs but not Omicron BA.1 RBD (Fig. 4H). Contrary to expectations, the population of B MEM cells exclusively reactive with Omicron BA.1 RBD was small in most of those individuals. We did not detect B MEM cells that exclusively recognized epitopes shared by both the Omicron BA.1 and Alpha RBDs or by the Omicron BA.1 and Delta RBDs.
Furthermore, in all individuals, we identified two additional subsets of RBD-specific B MEM cells (in bold in Fig. 4H). One subset was characterized by binding to the RBDs of Wuhan, Alpha, and Omicron BA.1 but not the Delta RBD. The other population exhibited binding to Wuhan and Alpha but not Omicron BA.1 or Delta RBD. Sequence alignment identified L452R as the only RBD alteration unique for Delta that is not shared by the other three variant RBDs (Fig. 4I,  top). Similarly, the only RBD site conserved in Wuhan and Alpha but altered in Delta and Omicron BA.1 was found to be T478K (Fig. 4I, bottom). Both L452R and T478K alterations are known to be associated with the evasion of vaccine-induced neutralizing antibody responses (31,32). Position L452 is mutated in the recently emerged SARS-CoV-2 Omicron sublineages BA.4 and BA.5 (33). Only minor B MEM cell frequencies were detected in those combinatorial subgroups in which multiple sequence alignment failed to identify unique epitopes in the RBD sequence (e.g., Wuhan only or Wuhan and Omicron BA.1 but not Alpha and Delta). These observations indicate that the B MEM cell response against RBD is driven by specificities induced through prior vaccination with BNT162b2 and not substantially redirected against variant-specific RBD epitopes displayed by the infecting Omicron BA.1 variant.

DISCUSSION
SARS-CoV-2 Omicron BA.1 is a partial immune escape variant with an unprecedented number of amino acid alterations in the S glycoprotein at sites of neutralizing antibody binding (15). Neutralizing antibody mapping and molecular modeling studies strongly support the functional relevance of these alterations (20,34), which is confirmed by the fact that double-vaccinated individuals have no detectable neutralizing activity against SARS-CoV-2 Omicron BA.1 (25,35).
In line with concurrently published reports (36,37), we show that Omicron BA.1 breakthrough infection of BNT162b2-vaccinated individuals augments broadly neutralizing activity against Omicron BA.1, BA.2, and previous VOCs to levels similar to those observed against the Wuhan strain. However, neutralization of the latest Omicron sublineages BA.4 and BA.5 was not enhanced, with titers comparable to those against the phylogenetically more distant SARS-CoV-1. Although our study focused on individuals vaccinated with the BNT162b2 mRNA vaccine, in individuals vaccinated with CoronaVac, similar observations suggest that Omicron BA.4/5 can bypass BA.1 infection-mediated boosting of humoral immunity (33).
Our study provides insights into how immunity against multiple variants is achieved and why Omicron BA.4 and BA.5 sublineages can partially escape neutralization. It suggests that initial exposure to the Wuhan strain S glycoprotein may have shaped the formation of B MEM cells and imprinted against novel B MEM cell responses recognizing epitopes distinctive for the Omicron BA.1 variant. Omicron BA.1 breakthrough infection in BNT162b2-vaccinated individuals primarily expands a broad B MEM cell repertoire against conserved S glycoprotein and RBD epitopes rather than inducing strictly Omicron BA.1-specific B MEM cells. Similar observations have been reported from vaccinated individuals who experienced breakthrough infections with the Delta variant and with the Omicron BA.1 sublineage (3,33).
As compared with the immune response induced by a homologous vaccine booster, an Omicron BA.1 breakthrough infection leads to a more substantial increase in antibody neutralization titers against Omicron and a robust cross-neutralization of many SARS CoV-2 variants. These effects are particularly notable in doublevaccinated individuals.
Three findings may point to potentially complementary and synergistic underlying mechanisms. The first is induction of broadly neutralizing antibodies. We found that most of the sera from Omicron BA.1 convalescent but not from Omicron-naïve vaccinated individuals robustly neutralize previous SARS-CoV-2 VOCs, including BA.1 and BA.2 and, to a far lesser extent, SARS-CoV-2 Omicron BA.4/5 and SARS-CoV-1. This indicates that Omicron BA.1 infection in vaccinated individuals stimulates B MEM cells that produce neutralizing antibodies against S glycoprotein epitopes conserved in the SARS-CoV-2 variants up to and including Omicron BA.2 but that have mostly been lost in BA.4/5 and are, for the most part, not shared by SARS-CoV-1. Over the past 2 years, broadly cross-neutralizing antibodies have been isolated from both SARS-CoV-2 and SARS-CoV-1 convalescent and/or vaccinated individuals (20,27,38) and are known to target highly conserved S glycoprotein domains (39,40). The greater antigenic distance of the Omicron BA.1 S glycoprotein from earlier SARS-CoV-2 strains may promote targeting of conserved subdominant neutralizing epitopes as recently described to be located, e.g., in cryptic sites within the RBD distinct from the receptor-binding motif (41,42) or in the membrane proximal S glycoprotein subunit designated S2 (43)(44)(45).
The second finding is a bias toward RBD-specific B MEM cell responses. Omicron BA.1-infected individuals appear to have a substantially higher RBD/S glycoprotein-specific B MEM cell ratio as compared with vaccinated Omicron-naïve individuals. Omicron BA.1 carries multiple S glycoprotein alterations such as del69/70 and del143 to del145 in key neutralizing antibody binding sites of the NTD that markedly reduce the targeting surface for B MEM cell responses in this region. Although the Omicron BA.1 RBD harbors multiple alterations, there are some unaffected neutralizing antibody binding sites left (20). An expansion of B MEM cells that produce neutralizing antibodies against RBD epitopes that are not altered in Omicron BA.1, such as those at position L452 as indicated in our study, could help to rapidly restore neutralization of the BA.1 and BA.2 variants. The strong neutralization of Omicron BA.1 and BA.2 should not mask the fact that the neutralizing B MEM immune response in Omicron BA.1 convalescent vaccinated individuals is driven by a smaller number of epitopes. The significantly reduced neutralizing activity against the Omicron BA.4/5 pseudovirus, which harbors the additional alterations L452R and F486V in the RBD, demonstrates the mechanism of immune evasion by the loss of the few remaining conserved epitopes. Meanwhile, further sublineages with L452 alterations (e.g., BA.2.12.1) are being reported to evade humoral immunity elicited by BA.1 breakthrough infection (33).
The third finding is an overall increase of S glycoprotein-specific B MEM cells. Omicron BA.1 convalescent double-vaccinated individuals appear to have a higher frequency of B MEM cells and higher neutralizing antibody titers against previous VOCs as compared with triple-vaccinated individuals. Studies on other VOCs have not shown breakthrough infections in double-vaccinated individuals to be superior to a third vaccine dose in eliciting neutralizing activity (4,36). This may be explained by poor neutralization of the partial escape Omicron BA.1 variant in the initial phase of infection, which may result in greater or prolonged antigen exposure of the immune system to the altered S glycoprotein.
In aggregate, our results suggest that despite potential imprinting of the immune response by previous vaccination, the preformed B cell memory pool can be refocused and quantitatively remodeled by exposure to heterologous S glycoproteins to allow the neutralization of variants that evade a previously established neutralizing antibody response. However, our data also suggest that the immunity in the early stage of Omicron BA.1 infection in vaccinated individuals is based on recognition of conserved epitopes and is narrowly focused on a small number of neutralizing sites that are not altered in Omicron BA.1 and BA.2. Such a narrow immune response bears a high risk that those few epitopes may be lost by acquisition of further alterations in the course of the ongoing evolution of Omicron and may result in immune escape, as is being experienced with sublineages BA.2.12.1, BA.4, and BA.5 (33,46). Omicron BA.1 breakthrough infection does not appear to reduce the overall spectrum of (Wuhan) S glycoprotein-specific B MEM cells, because B MEM cells that do not recognize Omicron BA.1 S remain detectable in blood at similar frequencies. We consistently detected Wuhan-specific (non-Omicron BA.1 reactive) B MEM cells in Omicron BA.1 breakthrough-infected individuals at levels similar to those in Omicron-naïve double-/triplevaccinated individuals. Our data therefore suggest an increase of the total B MEM cell repertoire by selective amplification of B MEM cells that recognize shared epitopes.
Our findings raise a number of questions, e.g., to what extent induced B MEM responses are functional and directed against neutralizing domains. A recent study examined more than 600 neutralizing antibodies isolated from triple-CoronaVac-vaccinated individuals who subsequently experienced BA.1 breakthrough infection. Consistent with our findings, the study showed that BA.1 infection in vaccinated individuals primarily retrieves Wuhan S glycoproteininduced B cell memory and elicits cross-reactive neutralizing antibodies against RBD epitopes that neutralize both the ancestral SARS-CoV-2 Wuhan and the Omicron BA.1 variant (33). In addition, it is not yet clear whether the B MEM cells against conserved epitopes that we observed after Omicron BA.1 breakthrough infection are newly recruited cross-reactive naïve B cells or rather expanded from the preexistent B MEM cell pool. A recent study investigating a third vaccine booster suggests that both mechanisms are relevant (47). Further, we cannot exclude that strictly Omicron-BA.1-specific B MEM cells are being efficiently generated but had just not been exported from the germinal center at the time point of our analysis. These questions can be addressed by comprehensive studies of the B cell repertoire at later time points (>3 months) after breakthrough infection, including BCR (B cell receptor) repertoire analysis by single-cell immunoglobulin (Ig) gene sequencing of antigen-specific B MEM cells, extended to the cloning, expression and characterization of monoclonal antibodies with regard to specificity, functional properties, and affinity.
Our findings are based on retrospective analyses of samples derived from different studies. Therefore, the sample sizes were relatively small, and cohorts were not fully adjusted with regard to immunization intervals, sampling time points, and demographic characteristics such as age and sex of individuals. Another limitation is that the analysis was restricted to B MEM cells; long-lived bone marrow-derived plasma cells, which are known to be BNT162b2 vaccination-induced (48), were not investigated because they cannot be cryopreserved.
A key motivation for our study was to inform our vaccine adaptation program. We expect that the currently ongoing vaccine adaptations to the Omicron BA.1 S glycoprotein, similar to the Omicron BA.1 breakthrough infection that we studied, may reshape the B cell memory repertoire and provide broad protection against previous VOCs. However, given the rapid evolution of SARS-CoV-2, other sublineages of Omicron that antigenically deviate from BA.1 even more than the immune escape variants BA.4/5 may have emerged by the time of potential authorization of those vaccines later this year. In a pandemic in which a highly transmissible VOC feeds dynamic and rapid evolution of altered variants, an effective strategy may be to leverage the full potential of mRNA vaccine technology, which allows production and release of new vaccines in less than 3 months. To enable adapted vaccines that truly reflect relevant VOCs at licensure, it would be prudent to build on decades of experience with seasonal influenza vaccines and implement timely, rapid licensure procedures that use the latest epidemiologic data to select COVID-19 vaccine strains.

Study design
The objective of this study was to investigate the effect of Omicron BA.1 breakthrough infection on the cross-variant neutralization capacity of human sera and how repeat SARS-CoV-2 antigen exposure modulates B MEM cell specificity in individuals vaccinated with BNT162b2. We compared immune responses in Omicron-naïve individuals double-or triple-vaccinated with BNT162b2 to those of individuals double-or triple-vaccinated with BNT162b2 with a confirmed subsequent breakthrough infection with Omicron during a period of Omicron sublineage BA.1 dominance. Serum neutralizing capability was characterized using live and pseudovirus neutralization assays, and flow cytometry was used to detect and characterize SARS-CoV-2specific B cells in bulk PBMCs. Cross neutralization of variants was further characterized in a cohort vaccinated with other approved COVID-19 vaccines or mixed regimens who experienced subsequent Omicron breakthrough infection. All participants had no documented history of SARS-CoV-2 infection before vaccination.
Participants from the SARS-CoV-2 Omicron convalescent doubleand triple-vaccinated cohorts (BNT162b2 2 + Omi and BNT162b2 3 + Omi cohorts, respectively) and individuals vaccinated with other approved COVID-19 vaccines or mixed regimens with subsequent Omicron breakthrough infection were recruited from the University Hospital, Goethe University Frankfurt as part of a research program that recruited patients who had experienced Omicron breakthrough infection after vaccination for COVID-19 to provide blood samples and clinical data for research. Omicron infections were confirmed with variant-specific polymerase chain reaction between November 2021 and mid-January 2022, at a time when sublineage BA.1 was dominant (24). The infections of seven participants in this study were further characterized by genome sequencing, five of whom were in the BNT162b2-vaccinated cohorts, and two in the cohort with participants vaccinated with other approved COVID-19 vaccines or mixed regimens. In all seven cases, genome sequencing confirmed Omicron BA.1 infection (tables S3 and S10).
Participants were free of symptoms at the time of blood collection. The study protocol for this research program was approved by the Ethics Board of the University Hospital, Goethe University Frankfurt (no. 2021-560). Demographic and clinical information for all participants as well as sampling time points are provided in tables S1 to S3 and S10 and Fig. 1. Serum was isolated by centrifugation 2000g for 10 min and cryopreserved until use. Li-heparin blood samples were isolated by density gradient centrifugation using Ficoll-Paque PLUS (Cytiva) and were subsequently cryopreserved until use.  (49).

Vesicular stomatitis virus-SARS-CoV-2 S variant pseudovirus generation
A diagram of SARS-CoV-2 S glycoprotein alterations is shown in fig. S7A. Briefly, human embryonic kidney-293T/17 monolayers [American Type Culture Collection (ATCC) CRL-11268] cultured in Dulbecco's modified Eagle's medium with GlutaMAX (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich) (referred to as medium) were transfected with Sanger sequencing-verified SARS-CoV-1 or variant-specific SARS-CoV-2 S expression plasmid with Lipofectamine LTX (Life Technologies) following the manufacturer's instructions. At 24 hours, VSV-G complemented VSVG vector. After incubation for 2 hours at 37°C with 7.5% CO 2 , cells were washed twice with phosphate-buffered saline (PBS) before medium supplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast Inc.) was added to neutralize residual VSV-Gcomplemented input virus. VSV-SARS-CoV-2-S pseudotypecontaining medium was harvested 20 hours after inoculation, passed through a 0.2-m filter (Nalgene), and stored at −80°C. The pseudovirus batches were titrated on Vero 76 cells (ATCC CRL-1587) cultured in medium. The relative luciferase units induced by a defined volume of a Wuhan S glycoprotein pseudovirus reference batch previously described by Muik et al. (26), which corresponds to an infectious titer of 200 transducing units (TUs) per milliliter, were used as a comparator. Input volumes for the SARS-CoV-2 variant pseudovirus batches were calculated to normalize the infectious titer on the basis of the relative luciferase units relative to the reference.

Pseudovirus neutralization assay
Vero 76 cells were seeded in 96-well, white, flat-bottom plates (Thermo Fisher Scientific) at 40,000 cells per well in medium 4 hours before the assay and cultured at 37°C with 7.5% CO 2 . Each serum was serially diluted twofold in medium with the first dilution being 1:5 (Omicronnaïve BNT162b2 double-and triple-vaccinated; dilution range of 1:5 to 1:5120) or 1:30 (BNt162b2 double-and triple-vaccinated after subsequent Omicron breakthrough infection; dilution range of 1:30 to 1:30,720). In the case of the SARS-CoV-1 pseudovirus assay, the sera of all individuals were initially diluted 1:5 (dilution range of 1:5 to 1:5120). VSV-SARS-CoV-2-S/VSV-SARS-CoV-1-S particles were diluted in medium to obtain 200 TUs in the assay. Serum dilutions were mixed 1:1 with pseudovirus (n = 2 technical replicates per serum per pseudovirus) for 30 min at room temperature before being added to Vero 76 cell monolayers and incubated at 37°C with 7.5% CO 2 for 24 hours. Supernatants were removed, and the cells were lysed with a luciferase reagent (Promega). Luminescence was recorded on a CLARIOstar Plus microplate reader (BMG Labtech), and neutralization titers were calculated as the reciprocal of the highest serum dilution that still resulted in 50% reduction in luminescence. Results were expressed as GMTs of duplicates. If no neutralization was observed, an arbitrary titer value of half of the limit of detection (LOD) was reported. Supplementary tables listing the neutralization titers are provided (tables S4 to S6 and S11).  /144/145, 211, L212I,  ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S,  S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H,  T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F) to allow any antigen-specific antibodies to bind to the virus. A diagram of S glycoprotein alterations is shown in fig. S7B. The 2019-nCOV/ITALY-INMI1 strain S glycoprotein is identical in sequence to the wild-type SARS-CoV-2 S (Wuhan-Hu-1 isolate). Vero E6 (ATCC CRL-1586) cell monolayers were inoculated with the serum/virus mix in 96-well plates and incubated for 3 days (2019-nCOV/ITALY-INMI1 strain) or 4 days (Beta, Delta, and Omicron BA.1 variant strain) to allow infection by non-neutralized virus. The plates were observed under an inverted light microscope, and the wells were scored as positive for SARS-CoV-2 infection (i.e., showing CPE) or negative for SARS-CoV-2 infection (i.e., cells were alive without CPE). The neutralization titer was determined as the reciprocal of the highest serum dilution that protected more than 50% of cells from CPE and reported as GMT of duplicates. If no neutralization was observed, an arbitrary titer value of 5 (half of the LOD) was reported. Supplementary tables listing the neutralization titers are provided (tables S7 to S9). Fixative (BD Biosciences, according to the manufacturer's instructions) before data acquisition on a BD Symphony A3 flow cytometer. FCS 3.0 files were exported from BD Diva Software and analyzed using FlowJo software (version 10.7.1.).

Statistical analysis
The statistical method of aggregation used for the analysis of antibody titers is the geometric mean and for the ratio of SARS-CoV-2 VOC titer and Wuhan titer, the geometric mean and the corresponding 95% confidence interval. The use of the geometric mean accounts for the non-normal distribution of antibody titers, which span several orders of magnitude. The Friedman test with Dunn's correction for multiple comparisons was used to conduct pairwise signed-rank tests of group geometric mean neutralizing antibody titers with a common control group. Flow cytometric frequencies were analyzed with and tables were exported from FlowJo software (version 10.7.1.). Statistical analysis of cumulative B MEM cell frequencies was the mean and SEM. Statistical significance was tested for using the nonparametric Friedman test with Dunn's multiple comparisons correction. All statistical analyses were performed using GraphPad Prism software version 9.