key: cord-0755396-5kz2s7ag
authors: Goel, Rishi R.; Painter, Mark M.; Lundgreen, Kendall A.; Apostolidis, Sokratis A.; Baxter, Amy E.; Giles, Josephine R.; Mathew, Divij; Pattekar, Ajinkya; Reynaldi, Arnold; Khoury, David S.; Gouma, Sigrid; Hicks, Philip; Dysinger, Sarah; Hicks, Amanda; Sharma, Harsh; Herring, Sarah; Korte, Scott; KC, Wumesh; Oldridge, Derek A.; Erickson, Rachel I.; Weirick, Madison E.; McAllister, Christopher M.; Awofolaju, Moses; Tanenbaum, Nicole; Dougherty, Jeanette; Long, Sherea; D’Andrea, Kurt; Hamilton, Jacob T.; McLaughlin, Maura; Williams, Justine C.; Adamski, Sharon; Kuthuru, Oliva; Drapeau, Elizabeth M.; Davenport, Miles P.; Hensley, Scott E.; Bates, Paul; Greenplate, Allison R.; Wherry, E. John
title: Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine
date: 2022-04-08
journal: Cell
DOI: 10.1016/j.cell.2022.04.009
sha: de4edb42fc6ffe9938742d968d1bc7df3adf3a62
doc_id: 755396
cord_uid: 5kz2s7ag

We examined antibody and memory B cell responses longitudinally for ∼9-10 months after primary 2-dose SARS-CoV-2 mRNA vaccination and 3 months after a 3rd dose. Antibody decay stabilized between 6 to 9 months and antibody quality continued to improve for at least 9 months after 2-dose vaccination. Spike- and RBD-specific memory B cells remained durable over time, and 40-50% of RBD-specific memory B cells simultaneously bound the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells were efficiently re-activated by a 3rd dose of wild-type vaccine and correlated with the corresponding increase in neutralizing antibody titers. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit the added protection afforded by repeat short interval boosting. These data provide insight into the quantity and quality of mRNA vaccine-induced immunity over time through 3 or more antigen exposures.

SARS-CoV-2 infections continue to cause significant morbidity and mortality worldwide (Carvalho et al., 2021) . Since the virus was identified in late 2019, several SARS-CoV-2 variants of concern (VOC) have emerged. Mutations found in SARS-CoV-2 variants, particularly those in the Spike glycoprotein, can alter viral transmission and immune recognition (Garcia-Beltran et al., 2021; Greaney et al., 2021a Greaney et al., , 2021b . Of these VOC, the Delta (B.1.617.2) variant had considerable impact due to its increased infectivity and partial escape from neutralizing antibodies (Mlcochova et al., 2021; Planas et al., 2021) . In November of 2021, scientists in South Africa identified and characterized the Omicron (B.1.1.529) variant (Viana et al., 2022) . In the weeks following identification, Omicron spread rapidly, outcompeting Delta to become the dominant variant in the US and many parts of the world.

A major concern about Omicron is the large number of mutations in the Spike protein, including ~15 amino acid changes in the Spike receptor binding domain (RBD). In vitro data indicate that these mutations have a substantial effect on evading antibody responses in convalescent or mRNA vaccinated (Pfizer BNT162b2 or Moderna mRNA-1273) individuals. This effect is more pronounced than other VOC, with a ~10 to ~40-fold reduction in neutralization capacity compared to wild-type virus using either pseudovirus or live virus neutralization assays, and little to no neutralizing activity against Omicron detected at >6 months after the primary 2-dose vaccine series (Cameroni et al., 2021; Cele et al., 2021; Garcia-Beltran et al., 2022; Schmidt et al., 2021a) .

In addition to circulating antibodies, memory B cells represent an important source of long-term immunity Victora and Nussenzweig, 2012) . In contrast to antibodies that decline over the first 3-6 months post-vaccination (Levin et al., 2021) , antigen-specific memory B cells appear highly stable over time (Goel et al., 2021a) . Upon re-exposure to antigen, either through vaccination or infection, these memory B cells can differentiate into antibody secreting cells (ASCs) and rapidly produce new antibodies (Laidlaw and Ellebedy, 2021) . Indeed, recent non-human primate studies of mRNA vaccination highlight recall antibody responses from memory B cells as a key factor in protection from severe COVID-19 pathology in the lungs (Gagne et al., 2022a) . Previous work has shown that mRNA vaccines induce robust memory B cell responses that continue to evolve via germinal center reactions for months after primary vaccination (Goel et al., 2021b (Goel et al., , 2021a Kim et al., 2021; Röltgen et al., 2022; Turner et al., 2021) .

J o u r n a l P r e -p r o o f 4 As a result, immunization with mRNA vaccines encoding the original Wuhan Spike protein generates a population of high-affinity memory B cells that can bind the Alpha, Beta, and Delta variants and produce neutralizing antibodies upon restimulation.

Serologic data indicate that antibody responses to Omicron can be at least partially boosted in the short-term (up to ~1 month) after a 3 rd vaccine dose (Muecksch et al., 2022; Muik et al., 2022; Schmidt et al., 2021b; Xia et al., 2022) , suggesting that immunological memory generated by 2dose vaccination has some reactivity against the Omicron Spike protein. A 3 rd vaccine dose also provides increased protection from Omicron variant infection (Shrestha et al., 2022) . However, it is unclear how long these boosted antibody responses to Omicron may last and what percent of memory B cells retain binding to Omicron and other variants. Moreover, the dynamics of memory B cell responses in humans are poorly understood, and whether boosting with the original Wuhan Spike can overcome antigenic changes by efficiently reactivating Omicron-binding memory B cells is unknown (Kotaki et al., 2022; Wang et al., 2022) . Finally, it remains unclear what features of immunity induced by 2-dose vaccination determine optimal boosting following a 3 rd vaccine dose, and how immune responses are affected by additional antigen encounters beyond a 3-dose vaccine schedule. The answers to these questions should inform how to optimize the use of additional vaccine doses for protection against Omicron and future VOC.

J o u r n a l P r e -p r o o f 5 Results:

We examined antibody and memory B cell responses to SARS-CoV-2 in a longitudinal cohort of 61 individuals receiving mRNA vaccines (Pfizer BNT162b2 or Moderna mRNA-1273) . This cohort has been previously described through 6 months post-2 doses of mRNA vaccine (Goel et al., 2021b (Goel et al., , 2021a Painter et al., 2021) . 45 individuals were infection naïve and 16 had recovered from a prior SARS-CoV-2 infection. Paired serum and peripheral blood mononuclear cell (PBMC) samples were collected at 10 different timepoints, ranging from pre-vaccine baseline through ~9-10 months post-primary 2-dose vaccination, as well as prior to a 3 rd vaccine dose, ~2 weeks post-3 rd dose, and ~3 months post-3 rd dose ( figure 1A) . Nine individuals had a confirmed postvaccination (commonly referred to as "breakthrough") infection during the study period and are indicated in all analyses. Additional cohort information is provided in tables S1-2.

As we have previously described for this cohort, 2-dose mRNA vaccination in previously uninfected individuals induced high titers of binding and neutralizing antibodies, whereas vaccination in individuals with a prior SARS-CoV-2 infection (commonly referred to as "hybrid immunity") resulted in higher antibody titers, consistent with an anamnestic response from prevaccination immunological memory (Goel et al., 2021b (Goel et al., , 2021a . Although these previous studies documented a decline in antibodies from their peak ~1 month post vaccination to 6 months, here we extended our analysis of this cohort to later time points. These data revealed a stabilization of antibody titers between 6-and 9-months post-vaccination for both individuals with and without previous SARS-CoV-2 infection, with little to no decrease in neutralizing antibody titers after 6 months (figure 1B-D). These findings are consistent with ongoing antibody production from longlived plasma cells in the later phases of immune memory after vaccination.

To evaluate the quality of antibody responses, we calculated an antibody potency index. Although antibody-mediated protection can be influenced by functions other than neutralization (Bournazos and Ravetch, 2017; Lu et al., 2017) , we defined a potency index based on the ratio of neutralization titers to the total concentration of anti-RBD binding IgG. Antibody potency increased significantly over time after the 2 nd vaccine dose, with a continued increase in potency from 6 to 9 months post-vaccination in the infection naïve group as antibody concentrations began to plateau ( figure   1E ). These observations suggest decay of lower quality antibody from short-lived antibody secreting cells, as well as continued emergence of post-germinal center affinity matured plasma J o u r n a l P r e -p r o o f 6 cells over time that produce higher quality antibody later in the response. This improvement in the quality of antibody for at least 9 months is also consistent with a recent report demonstrating the continued presence of Spike-binding germinal center B cells in axillary lymph nodes at 29 weeks post-vaccination .

In addition to the primary 2-dose vaccine series, most of our cohort went on to receive a 3 rd dose of mRNA vaccine. A 3 rd dose of vaccine in infection naïve individuals significantly increased binding and neutralizing antibodies, with both reaching a similar level to that observed in previously infected individuals after the 2-dose vaccine series ( figure 1B-D) . A 3 rd dose of mRNA vaccine in these COVID recovered individuals (i.e. a 4 th exposure) also significantly boosted antibody responses; however, the relative magnitude of this increase was less than observed in the recall response after the initial 2-dose vaccine series (figure 1B-D). Several individuals in this cohort also experienced breakthrough infections after 2 or 3 doses of vaccine. Although the sample size was limited (N=3), infection following 2-dose vaccination appeared to boost antibodies to similar levels compared to previous infection with 2 doses of vaccine ( figure 1B-D) ,

suggesting that the total number of antigen exposures may be as important as the relative order of exposure to infection and vaccination.

To quantify neutralizing capacity of vaccine-induced antibody responses against VOC, we generated pseudotyped viruses encoding the Delta and Omicron Spike proteins. Consistent with previous reports, neutralizing titers against Omicron were significantly reduced relative to D614G, with ~20% of individuals having Omicron neutralization titers below the limit of detection at ~9 months post-primary vaccination (figure 1F-G). Following a 3 rd dose, neutralizing titers to Omicron increased by a median of ~30-fold, with similar kinetics and magnitude of boosting as neutralizing antibodies against D614G (figure 1F-G). Although neutralization against Omicron declined 1.8-fold from peak levels between 2 weeks post-3 rd dose and 3 months post-3 rd dose, titers remained 15-fold above pre-3 rd dose baseline in COVID-naïve individuals (figure 1F),

indicating that an additional vaccine dose has a lasting benefit for antibodies against Omicron. In paired comparisons for individuals, Omicron neutralizing antibodies had a 6.4-fold lower median neutralizing titer compared to D614G at the peak response after the 3 rd vaccine dose and 3.8-fold lower titer 3 months later. Despite the relative loss of neutralizing activity, peak Omicron neutralizing titers after the 3 rd dose were comparable to neutralizing titers against D614G ~1 week after the 2 nd vaccine dose, where clinical efficacy has previously been defined (figure 1F-G) J o u r n a l P r e -p r o o f 7 . Moreover, Omicron neutralizing titers at 3 months post-3 rd dose were higher than pre-3 rd dose D614G neutralizing titers (figure 1F-G).

Finally, to investigate potential differences in the quality of antibody recall responses, we compared antibody potency 2 weeks after the first dose of mRNA vaccine in individuals with preexisting immune memory from infection to antibody potency 2 weeks after a 3 rd dose of mRNA vaccine in previously uninfected individuals. Previous reports have suggested that infection generates greater antibody potency and breadth than 2-dose mRNA vaccination alone (29), but little is known about the impact of a 3rd vaccine dose on antibody potency. In this cohort, antibody potency against both D614G and Omicron was slightly higher following a 3 rd dose of mRNA vaccine compared to recall responses following the 1 st dose in SARS-CoV-2 recovered individuals (figure 1H-I), suggesting that a 3 rd dose of mRNA vaccine drives antibody potency to similar levels to "hybrid immunity". Of note, potency continued to increase in SARS-CoV-2 recovered individuals between the initial recall response to vaccine and ~9 months post-vaccination (figure 1E,H-I).

This finding indicates there may be ongoing evolution after a vaccine-induced recall response that can result in further improvement of antibody potency. Taken together, these data demonstrate that antibody responses, including neutralizing antibodies to Omicron, are effectively boosted by a 3 rd dose of mRNA vaccine with sustained benefit at ~3 months post-3 rd dose.

We next investigated B cell responses to mRNA vaccination. Antigen-specific B cell responses were quantified from bulk PBMCs by flow cytometry using fluorescently labelled SARS-CoV-2 Spike and RBD probes as previously described Goel et al., 2021b Goel et al., , 2021a .

Influenza Hemagglutinin (HA) was used as a historical antigen for a specificity control.

Plasmablasts were identified as CD20-CD38++ non-naïve B cells. Memory B cells were identified as CD20+ CD38lo/int non-naïve B cells. Full gating strategies are shown in figure S1.

Consistent with our plasma antibody data, re-exposure to SARS-CoV-2 antigen, either through a 3 rd mRNA vaccine dose or breakthrough infection, resulted in significant expansion of Spikebinding plasmablasts ~1 week after antigen encounter ( figure 2A-B) . Overall, the rapid emergence of antibody-secreting cells following antigen re-encounter is consistent with recall from a pool of memory B cells. These data are also consistent with findings after viral challenge in SARS-CoV-2 mRNA-vaccinated monkeys, where anamnestic antibody responses from memory B cells were identified as a major protective mechanism (Gagne et al., 2022a) . J o u r n a l P r e -p r o o f 8 We previously demonstrated that mRNA vaccines induce durable and functional memory B cells to SARS-CoV-2 that are stable for at least 6 months after vaccination (Goel et al., 2021a) . Here, we extended these observations by tracking responses further into the memory phase. Spikeand RBD-specific memory B cell numbers continued to remain highly stable through at least 9 months post-vaccination in both SARS-CoV-2 naïve and previously infected individuals with no evidence of decline in numbers from 6 to 9 months post-primary vaccination (figure 2C-E).

Notably, 34/35 SARS-CoV-2 naïve individuals had Spike-and RBD-specific memory B cell frequencies above their pre-vaccine baseline at the 9-month timepoint, highlighting the continued durability of mRNA-vaccine induced cellular immunity.

Upon receipt of a 3 rd mRNA vaccine dose, these memory B cells expanded in number. At ~2 weeks post-boost there was a median 2.2-fold fold increase in Spike-specific and 3.6-fold increase in RBD-specific memory B cells in infection naïve individuals, with similar boosting for Spike-and RBD-specific memory B cells in COVID recovered vaccinees (figure 2F-G). By 3 months post 3 rd vaccine dose in infection naïve subjects, memory B cells had declined from peak levels but still remained ~1.5-fold more abundant than before boosting (figure 2F-G). A 3 rd vaccination did not markedly change the isotype composition of the response from what was reported previously in this cohort after two doses, with a majority of memory B cells remaining IgG+ (figure 2H). A 3 rd dose of vaccine also induced a population of CD71+ activated B cells, consistent with reactivation of memory B cells ( figure 2I ). This activation status, however, transitioned back to a resting memory phenotype by 3 months post-3 rd dose (figure 2I). Thus, memory B cells were rapidly reactivated by re-exposure to antigen, either through infection or vaccination, and this reactivation was associated with induction of plasmablasts, numerical expansion of memory B cells, and re-establishment of B cell memory.

A major question is whether vaccination with the original Wuhan Spike protein induces effective immunological memory to VOC including Omicron, and if so, whether Omicron-specific memory total PBMCs by negative selection at 3 timepoints: pre-3 rd dose, ~2 weeks after the 3 rd dose, and ~3 months after the 3 rd dose. Representative plots and gating strategy are shown in figure 3A-B and figure S2. Briefly, antigen-specificity and phenotype were identified according to the following gating strategy: Spike+ memory B cells were first identified from total memory B cells as described above. Spike+ memory B cells were subsequently gated based on co-binding to NTD or S2 probes. Memory B cells that were Spike+ but did not bind NTD or S2 were then examined for binding to the WT RBD probe, as well as variant RBD probes.

As previously described, 2-dose vaccination induced memory B cells specific for all domains of the Spike protein, with S2 representing the immunodominant part of the response (Goel et al., 2021a) . A 3 rd dose of mRNA vaccine boosted NTD-, WT RBD-and S2-specific memory B cells with a 2.4-fold increase in NTD-specific memory B cells, 3.2-fold increase in WT RBD-specific memory B cells, and a 1.9-fold increase in S2-specific memory B cells ( figure 3C-D) . Notably, memory B cells that recognized all RBD variants (Alpha, Beta, Delta, and Omicron) simultaneously had the greatest fold change after the boost (3.8-fold; figure 3C-D). All RBDbinding memory B cells, regardless of cross-binding specificity, declined in frequency from peak levels to 3 months after the 3 rd dose, but these memory B cells remained above pre-boost levels in most individuals (figure 3D). Taken together, these data indicate that a 3 rd exposure to wildtype Spike was sufficient to expand memory B cells targeting multiple different VOC.

To further investigate variant reactivity within the memory B cell compartment, we quantified cross-binding to different VOC probes as a percentage of WT RBD-binding cells. Nine months after primary vaccination, >90% of memory B cells that bound WT RBD also bound Alpha RBD containing a single N501Y substitution (figure 3E). The L452R and T478K mutations found in Delta resulted in moderate loss of binding, with ~80% of WT RBD+ cells still able to cross-bind Delta RBD (figure 3E). The K417N, E484K, and N501Y mutations found in Beta were slightly more immune evasive than Delta, with ~70% of WT RBD+ memory B cells able to cross-bind Beta RBD ( figure 3E) . Notably, ~55% of WT RBD-binding memory B cells after 2 doses of vaccine were still able to cross-bind Omicron RBD (figure 3E). Most Omicron RBD-specific memory B cells were also capable of recognizing Alpha, Beta and Delta RBDs, though binding overlap was less complete for Omicron and Delta compared to other combinations (figure 3F), likely due to the L452R mutation that is found in Delta but not Omicron. As a result, a considerable fraction J o u r n a l P r e -p r o o f 10 (~40-50%) of RBD-specific memory B cells could bind all 4 VOC RBDs simultaneously (figure 3E-F). These "All Variant+" memory B cells may represent a source of broad protection that is resilient to future VOC. Boosting appeared to slightly increase memory B cell cross-binding to Beta, Delta, and Omicron RBDs (figure 3E), and the overall VOC binding profiles were similar in both COVID-naïve and prior COVID vaccinees pre-and post-boost (figure 3G).

To investigate how memory B cells with different antigen specificities were impacted by a 3 rd dose of vaccine containing Wuhan Spike, we examined the activation phenotype of these cells ~2 weeks after the 3 rd vaccine dose. Memory B cell activation state was defined based on expression of CD21, CD27, and CD11c (figure 3B). CD21-CD27+ B cells were identified as activated memory (AM) and CD21+ CD27+ cells were identified as resting memory (RM) (Lau et al., 2017) . Taken together, these data indicate that mRNA vaccines encoding the original Wuhan Spike protein generated memory B cells that bind Omicron and other variant RBDs. These memory B cells were maintained without decline for at least 9-10 months after the primary 2-dose vaccine series. A 3 rd dose of mRNA vaccination efficiently activated Omicron-specific memory B cells at J o u r n a l P r e -p r o o f a similar proportion to Omicron RBD non-binding memory B cells. Thus, mRNA vaccination generates a robust population of memory B cells that maintain reactivity against multiple SARS-CoV-2 VOC, including Omicron, and these cells are efficiently re-engaged by a 3 rd vaccine dose.

Having quantified antibody and memory B cell responses individually, we next evaluated relationships between different antigen-specific antibody and memory B cell parameters over the course of primary 2-dose vaccination and after a 3 rd vaccine dose. To visualize the trajectory of vaccine-induced immunity over time, we clustered samples based on antibody and memory B cell responses using uniform manifold approximation and projection (UMAP) (figure 5A). Infectionnaïve and COVID-recovered individuals clustered apart from each other at the pre-vaccination baseline timepoint, as well as at early timepoints following the primary 2-dose vaccine series (figure 5B). Notably, these two groups began to converge in UMAP space at later memory timepoints and were indistinguishable after the 3 rd vaccine dose. This was also true in an additional UMAP generated using a larger set of parameters at pre-and post-boost timepoints, including variant-specific memory B cells, Omicron neutralizing antibodies, and memory B cell phenotype ( figure S3A-B) . We also examined correlations between antibody and memory B cell responses over time. Correlation analysis was restricted to individuals without prior COVID or post-vaccine infection. Antibody and memory B cell responses became more tightly correlated at memory timepoints before the 3 rd dose (figure 5C, figure S3C), supporting other findings that mRNA vaccines generate coordinated germinal center responses that ultimately result in the export of both long-lived plasma cells and memory B cells . Finally, we investigated how different immune features and sequential exposures to SARS-CoV-2 Spike protein affected the absolute and relative magnitude of recall responses. The pre-3 rd dose frequency of RBD+ memory B cells correlated with the absolute change in neutralizing antibody titers (pre-3 rd dose subtracted from post-3 rd dose) against D614G and Omicron ~2 weeks after revaccination ( figure 5D, figure S3D ). This observation is consistent with the notion that memory B cells generated after 2 doses of vaccine are an important predictor of subsequent recall responses to SARS-CoV-2 antigens. When comparing individuals with 3 exposures to SARS-CoV-2 Spike antigen (three vaccine doses) versus 4 (infection plus three vaccine doses), both groups showed a similar increase in antibody levels after the 3 rd vaccine dose (figure 5E). The absolute increase in antibody titer after the 3 rd vaccine dose was not obviously correlated with antibody titers prior to the 3 rd vaccine dose, suggesting that the frequency of RBD-binding memory J o u r n a l P r e -p r o o f B cells prior to boosting is the primary determinant of the magnitude of new antibody production (figure 5F). Individuals reached similar peak anti-Spike-and RBD-binding antibody titers after the 3 rd vaccine dose regardless of whether it was their 3 rd or 4 th exposure (figure 5G). Despite similar binding antibody levels, individuals with 4 total exposures reached slightly higher neutralizing titers against D614G and Omicron (figure 5G). Pre-3 rd dose antibody titers were moderately correlated with peak post-3 rd dose antibody titers ( figure 5H, figure S3C ), suggesting that antibody production after boosting is additive across the full range of pre-boost antibody levels.

To determine whether residual antibody levels affected the relative benefit of a 3 rd vaccine dose, we calculated the fold change in binding and neutralizing antibodies at 2 weeks after revaccination compared to paired pre-3 rd dose samples. A 3 rd vaccine dose induced a 10-100-fold increase in antibody titers for individuals with 3 total immune exposures to SARS-CoV-2 Spike (figure 5I). By contrast, the fold increase in antibodies following a 3 rd vaccine dose in those with previous COVID (i.e., 4 total exposures) was significantly lower with only a 5-10 fold-boost ( figure   5I ). Notably, the pre-3 rd dose concentration of anti-Spike or anti-RBD IgG was strongly negatively correlated with the corresponding fold change in antibody after the 3 rd vaccine dose regardless of exposure history (figure 5J). Accordingly, the relative benefit of boosting with an additional vaccine dose may be greatest for individuals with lower levels of pre-boost antibody.

One concern that has arisen is that vaccinating too soon after a previous exposure might lead to limited boosting. Existing data on this topic for SARS-CoV-2 are so far limited to the time interval between the 1 st and 2 nd vaccine doses (Chatterjee et al., 2022) . Here, we did not observe any significant association between peak antibody titers post-3 rd dose and time since primary 2 dose vaccination (figure S3E; range = 206-372 days), though there was a weak positive association between the fold-change in antibody responses after a 3 rd dose and the time since primary vaccination (figure S3F). Taken together, these data suggest that boosting with a 3 rd dose of mRNA vaccine efficiently re-engages memory B cells to produce new antibodies. Moreover, despite an increase in antibody titers in all subjects after the 3 rd vaccine dose, the relative benefit of this increase, measured by fold-change in antibody titers, was greatest in those with lower preboost antibody levels.

J o u r n a l P r e -p r o o f 13 Discussion:

mRNA vaccination generates protective immunity against SARS-CoV-2 by inducing potent antibody responses as well as memory B cells that can rapidly respond and produce new antibodies upon antigen re-exposure. The establishment of immunity after 2 doses of mRNA vaccine has been well characterized Goel et al., 2021a; Mateus et al., 2021; Rodda et al., 2022; Tarke et al., 2022) . However, it remains unclear how additional vaccine doses and combinations of vaccination and infection affect the magnitude and quality of immune responses, particularly against immune-evasive SARS-CoV-2 variants like Omicron. In this study, we examined antibody and memory B cell responses through ~9 months following the primary 2dose vaccine series as well as up to ~3 months following a 3 rd vaccine dose. In particular, the longitudinal nature of this study enabled detailed analysis of the magnitude, durability, and quality of SARS-CoV-2 vaccine-induced immunity over ~1 year and multiple antigen exposures.

This study provides several key pieces of data relevant to SARS-CoV-2 and mRNA vaccine immunobiology. Although antibody titers declined from peak levels observed ~1 week after the second dose to ~6 months post-primary vaccination, these antibody titers then stabilized between 6-and 9-months post-vaccination with continued improvement of neutralization potency over this period. A 3 rd vaccine dose at ~9 months post-primary vaccination increased antibody responses ~10 to 100-fold, including boosting neutralizing antibodies against the Omicron variant. Moreover, the antibody titers achieved after the 3 rd dose were similar to those observed in SARS-CoV-2 recovered individuals after 2 doses of mRNA vaccine, commonly referred to as hybrid immunity.

Breakthrough infection after 2 doses of mRNA vaccine also appeared to produce similar increases in antibody to a 3 rd vaccine dose. These boosted antibody responses subsequently declined over time but still remained significantly above pre-boost levels at 3 months post-3 rd dose.

In contrast to antibodies, which decayed over time following vaccination, memory B cell numbers remained highly stable in the blood with no evidence of decay at ~9 months post-primary vaccination. Notably, 2-dose vaccination generated a robust memory B cell response against the Omicron variant, with ~40-50% of RBD-binding memory B cells able to cross-bind Alpha, Beta, Delta, and Omicron. A 3 rd vaccine dose efficiently recruited memory B cells with cross-reactivity to multiple VOC, resulting in amplification of antibody responses capable of neutralizing Spike proteins from immune-evasive SARS-CoV-2 VOC including Omicron. The ability of approximately half of the memory B cell pool to bind multiple variants indicates that the antibodies encoded by J o u r n a l P r e -p r o o f 14 these memory B cells are targeting more conserved epitopes of RBD or are of higher quality and able to overcome epitope changes associated with mutations in VOC. It will be important to determine how resilient these memory B cell responses are to emerging variants. For example, BA.2 shares many features with previous VOC but also contains additional mutations not observed in the BA.1 Omicron sublineage (Yamasoba et al., 2022) . Regardless, mRNA vaccines encoding the original Wuhan Spike are capable of generating and boosting memory B cell responses and associated antibodies with the capacity to recognize major current SARS-CoV-2 VOC and may provide lasting protection against future variants.

Our data also identify several factors that predict the absolute magnitude and relative benefit of boosting. Boosting with a 3 rd vaccine dose universally increased binding and neutralizing antibody responses compared to pre-boost levels, and pre-boost memory B cell frequency was the best predictor of the increase in antibody levels after boosting. Binding antibody titers achieved after a 15 may augment recall responses compared to boosting with the original Wuhan strain sequence (Gagne et al., 2022b) . Nevertheless, these data highlight the urgent need to better understand what antibody titers are necessary for protection against infection and/or severe disease . Should this threshold be defined, it may be useful to implement serologic testing to maximize the benefit and equity of additional vaccine doses moving forward.

There are several limitations to this study. Although we studied immunological responses to mRNA vaccination, the precise correlates of vaccine efficacy are still being defined. Thus, these data on antibody and memory B cell responses cannot be directly translated to levels of clinical protection. In addition, we measured antibody quality using an index dependent on neutralization, but did not measure other non-neutralizing functions of antibodies that could have additional roles in antiviral immunity. Regarding our investigation of memory B cells, our strategy for examining Omicron RBD-reactive cells required simultaneous binding to WT RBD. It remains possible that there are also populations of Omicron RBD-specific memory B cells that do not bind WT RBD.

Finally, this study examined predominantly young, healthy subjects, and the immunological responses observed may not be entirely representative of those observed in older individuals, those with compromised immune systems, or other demographically distinct cohorts.

J o u r n a l P r e -p r o o f 16 Acknowledgements: We thank the study participants for their generosity in making the study possible. We also thank Ali Ellebedy and Julian Zhou for helpful discussions and feedback, as well as the Flow Cytometry Core at the University of Pennsylvania for technical support. indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Binding antibody and D614G pseudovirus neutralization data from pre-vaccine baseline through six months postprimary vaccination were described previously (Goel et al., 2021a) . 19 **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. See also Figure S1 . Memory B cell responses from pre-vaccine baseline through six months post-primary vaccination were reanalyzed from a previous dataset (Goel et al., 2021a) . vaccination. For B-D, analysis was restricted to SARS-CoV-2 naïve vaccinees. Statistics were calculated using paired non-parametric Wilcoxon test with Benjamini-Hochberg correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. See also Figure S2 . 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, E. John Wherry (wherry@pennmedicine.upenn.edu).

This study did not generate new unique reagents.

• All data reported in this paper will be shared by the lead contact upon request.

• This paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Sixty-one individuals were enrolled in the longitudinal vaccine study with informed consent and approval from the University of Pennsylvania Institutional Review Board (IRB# 844642). Of the 61 individuals, 45 were SARS-CoV-2 naïve prior to receiving their first vaccine dose and 16 had recovered from prior SARS-CoV-2 infection. All participants were otherwise healthy, with no selfreported history of chronic health conditions. Prior SARS-CoV-2 infection status was determined by a combination of self-reporting and laboratory detection of pre-existing immune responses. All subjects received mRNA vaccines, either Pfizer (BNT162b2) or Moderna (mRNA-1273). Samples were collected at 10 timepoints: baseline (T1), 2 weeks post-1 st dose (T2), day of 2 nd dose (T3), 1 week post-2 nd dose (T4), 3 months post-primary immunization (T5), 6 months post-primary immunization (T6), 9-10 months post-primary immunization (T7), pre 3 rd dose (T8), 2 weeks post 3 rd dose (T9), and 3 months post 3 rd dose (T10). Individuals received booster doses between 6 and 12 months post-primary vaccination. For clarity, we assigned booster doses as taking place at 9 months on longitudinal graphs. For the subset of individuals who received a booster vaccination after collection of T7, an additional sample was collected when they were transferred to the booster sub-study (T8). For those who received a booster dose before the T7 timepoint J o u r n a l P r e -p r o o f 24 was collected, they were transferred to the booster sub-study for collection of T8 without collection of T7. For simplicity, T7 and T8 were visualized as a single timepoint at ~9 months in most figures.

Peripheral blood samples (80-100mL) and clinical questionnaire data were collected at each of the timepoints listed above. Full cohort and demographic information is provided in table S1.

Additional healthy donor PBMC samples were collected with approval from the University of Pennsylvania Institutional Review Board (IRB# 845061).

Venous blood was collected into sodium heparin and EDTA tubes by standard phlebotomy.

Plasma was separated by centrifuging blood tubes at 3000rpm for 15 minutes and aliquots were stored at -80°C for subsequent antibody analyses. The remaining cellular fraction was diluted with an equal volume of RPMI + 1% FBS + 2mM L-Glutamine + 100 U Penicillin/Streptomycin (R10 medium) and layered above a lymphoprep gradient (STEMCELL Technologies) in SEPMATE tubes (STEMCELL Technologies). SEPMATE tubes were centrifuged at 1200g for 10 minutes and the PBMC fraction was decanted into fresh tubes, washed once with R10, and treated with ACK lysis buffer (Thermo Fisher) for 5 minutes. The ACK reaction was stopped with an additional wash, and cells were then resuspended in R10, filtered with a 70µm cell strainer, and counted using a Countess automated cell counter (Thermo Fisher). PBMCs were aliquoted and cryopreserved in 90% FBS 10% DMSO.

SARS-CoV-2-specific antibody levels in plasma were measured by enzyme-linked immunosorbent assay (ELISA) as previously described (Flannery et al., 2020; Goel et al., 2021b Goel et al., , 2021a . Plasmids encoding recombinant full-length SARS-CoV-2 Spike protein and RBD were provided by F. Krammer (Mt. Sinai) and purified by nickel-nitrilotriacetic acid resin (Qiagen).

ELISA plates (Immulon 4 HBX, Thermo Fisher Scientific) were coated overnight at 4°C with 2 g/mL recombinant protein or PBS. After overnight incubation, the plates were washed with phosphate-buffered saline containing 0.1% Tween-20 (PBS-T) and then blocked for 1 hour with PBS-T supplemented with 3% non-fat milk powder. Plasma samples were heat-inactivated for 1 hour at 56°C and diluted in PBS-T supplemented with 1% non-fat milk powder. After washing the plates with PBS-T, 50 L of diluted sample was added to each well and plates were incubated for 2 hours. Plates were washed again with PBS-T and then incubated for 1 hour with 50 L of 1:5000 J o u r n a l P r e -p r o o f 25 diluted goat anti-human IgG-HRP (Jackson ImmunoResearch Laboratories) or 1:1000 diluted goat anti-human IgM-HRP (SouthernBiotech). After secondary antibody, plates were washed again with PBS-T. 50 L SureBlue 3,3',5,5'-tetramethylbenzidine substrate (KPL) was added to each well and plates were incubated for 5 minutes. The reaction was then stopped by adding 25 L of 250 mM hydrochloric acid to each well. Plates were read with a SpectraMax 190 microplate reader (Molecular Devices) at an optical density (OD) of 450 nm. Monoclonal antibody CR3022 was included on each plate to convert OD values into relative antibody concentrations. Plasmids to express CR3022 were provided by I. Wilson (Scripps).

HEK 293T cells were seeded at 5 X 10 6 cells per 10 cm dish, incubated for 24 hours, and Before performing the neutralization assay, serum samples were thawed and heat-inactivated for 30 minutes at 53 ⁰C. Vero E6 cells stably expressing TMPRSS2 were seeded at a density of 2.5x10 4 cells/well in 100 l in a 96 well collagen-coated plate. VSVΔG-RFP SARS-CoV-2 pseudotype virus (100-300 focus forming units/well) was mixed with serum samples from a serial two-fold dilution and incubated for 1 hour at 37⁰C. To neutralize any potential VSV-G carryover virus, mouse anti-VSV Indiana G antibody (1E9F9) was also added at a concentration of 600 ng/ml (Absolute Antibody, Ab01402-2.0), and the VeroE6 TMPRSS2 cell culture media was replaced with this serum-virus mixture. After 21-22 hours, VeroE6 cells were washed and fixed with 4% paraformaldehyde before visualization on an S6 FluoroSpot Analyzer (CTL, Shaker Heights OH). Individual infected foci were enumerated and the values were compared to control wells without antibody. The focus reduction neutralization titer 50% (FRNT50) was measured as the greatest serum dilution at which focus count was reduced by at least 50% relative to control J o u r n a l P r e -p r o o f 26 cells that were infected in the absence of human serum. The geometric mean FRNT50 titers for each sample were reported based on readings from at least two technical replicates. 

Antigen-specific B cells were detected as previously described (Goel et al., 2021b (Goel et al., , 2021a . anti-IgA for 30 minutes at 4°C. Cells were then washed again and fixed overnight at 4°C using 1% PFA in PBS. Gates were set using healthy donor samples stained without antigen probes and identical gates were used for all experimental runs.

Variant RBD, NTD, and S2-specific memory B cells were measured using an approach similar to what was used to measure WT Spike antigens above. In this assay, SARS-CoV-2 nucleocapsid was used as a control antigen specific for infection but not vaccination. Probes were multimerized individually at 4°C for 1.5 hours by combing biotinylated proteins and fluorophore-conjugated SA at ~4:1 molar ratios (moles of SA calculated without considering the fluorophore): 200ng fulllength Spike protein was incubated with 20ng SA-BV421, 30ng N-terminal domain was incubated with 12ng SA-BV786, 25ng wild-type RBD was incubated with 12.5ng SA-BB515, 25ng Alpha RBD was incubated with 12.5ng SA-BV711, 25ng Beta RBD was incubated with 12.5ng SA-PE, 25ng Delta RBD was incubated with 12.5ng SA-APC, 25ng Omicron RBD was incubated with 12.5ng SA-PE-Cy7, 50ng S2 was incubated with 12ng SA-BUV737, 50ng nucleocapsid was incubated with 14ng SA-BV605. 12.5ng SA-BUV615 was used as a decoy probe. Antigen probes were prepared fresh before each stain and then pooled in together as an antigen probe master 

Flow cytometry data was collected on a BD Symphony A5 instrument. Standardized SPHERO rainbow beads (Spherotech) were used to track and adjust photomultiplier tubes over time.

UltraComp eBeads (Thermo Fisher) were used for compensation. Up to 5x10 6 cells were acquired per sample. Data were analyzed using FlowJo v10 (BD Bioscience). For Boolean analysis of J o u r n a l P r e -p r o o f 28 variant cross-binding, data were imported into SPICE 6 (NIH Vaccine Research Center (Roederer et al., 2011) ).

All data were analyzed in RStudio using custom scripts. Heatmaps were generated using the pheatmap package in R. Pairwise non-parametric correlations between variables were calculated and visualized as a correlogram using corrplot with FDR correction as described previously (Mathew et al., 2020) . Five antigen-specific immune features were used to train the UMAP in ** ** ** ** ** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** 

Fcγ Receptor Function and the Design of Vaccination Strategies

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift

The first 12 months of COVID-19: a timeline of immunological insights

Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization

SARS-CoV-2 Omicron Spike recognition by plasma from individuals receiving BNT162b2 mRNA vaccination with a 16-weeks interval between doses

Anti-SARS-CoV-2 receptor-binding domain antibody evolution after mRNA vaccination

Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection

SARS-CoV-2 seroprevalence among parturient women in Philadelphia

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in rhesus macaques coincides with anamnestic antibody response in the lung

mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits comparable B cell expansion, neutralizing antibodies and protection against Omicron

Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity

mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant

mRNA vaccines induce durable immune memory to SARS-CoV-2 and variants of concern

Distinct antibody and memory B cell responses in SARS-CoV-2 naïve and recovered individuals after mRNA vaccination

Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies

Mapping mutations to the SARS-CoV-2 RBD that escape binding by different classes of antibodies

Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection

Germinal centre-driven maturation of B cell response to SARS-CoV-2 vaccination

SARS-CoV-2 Omicron-neutralizing memory Bcells are elicited by two doses of BNT162b2 mRNA vaccine

The germinal centre B cell response to SARS-CoV-2

Low CD21 expression defines a population of recent germinal center graduates primed for plasma cell differentiation

Waning Immune Humoral Response to BNT162b2 Covid-19 Vaccine over 6 Months

Beyond binding: antibody effector functions in infectious diseases

Low dose mRNA-1273 COVID-19 vaccine generates durable T cell memory and antibodies enhanced by pre-existing crossreactive T cell memory

Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

Increased Potency and Breadth of SARS-CoV-2 Neutralizing Antibodies After a Third mRNA Vaccine Dose

Neutralization of SARS-CoV-2 Omicron by BNT162b2 mRNA vaccine-elicited human sera

Rapid induction of antigen-specific CD4+ T cells is associated with coordinated humoral and cellular immune responses to SARS-CoV-2 mRNA vaccination

Different B cell populations mediate early and late memory during an endogenous immune response

Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization

Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity

SPICE: Exploration and analysis of postcytometric complex multivariate datasets

Immune imprinting, breadth of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination

Plasma Neutralization of the SARS-CoV-2 Omicron Variant

High genetic barrier to SARS-CoV-2 polyclonal neutralizing antibody escape

Adaptive immunity to SARS-CoV-2 and COVID-19

COVID-19) Vaccine Boosting in Persons Already Protected by Natural or Vaccine-Induced Immunity

SARS-CoV-2 vaccination induces immunological T cell memory able to cross-recognize variants from Alpha to Omicron

SARS-CoV-2 mRNA vaccines induce persistent human germinal centre responses

Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa

Germinal centers

Memory B cell repertoire from triple vaccinees against diverse SARS-CoV-2 variants

Neutralization and durability of 2 or 3 doses of the BNT162b2 vaccine against Omicron SARS-CoV-2

Virological characteristics of SARS-CoV-2 BA.2 variant

serum_spike_IgG.x