Imprinted antibody responses against SARS-CoV-2 Omicron sublineages

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages carry distinct spike mutations resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters elicit plasma-neutralizing antibodies against Omicron BA.1, BA.2, BA.2.12.1, and BA.4/5, and that breakthrough infections, but not vaccination alone, induce neutralizing antibodies in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1, BA.2, and BA.4/5 receptor-binding domains, whereas Omicron primary infections elicit B cells of narrow specificity up to 6 months after infection. Although most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant–neutralizing antibody that is a strong candidate for clinical development. Description Defending against Omicron The Omicron BA.1 lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2021 and quickly became dominant, in part because of a large number of mutations that allowed escape from existing antibodies. New infection waves have come from other Omicron sublineages. Park et al. found that either a vaccination booster or a breakthrough infection elicits neutralization activity against the Omicron variants, but only a breakthrough infection induces an antibody response in the nasal mucosa, which might give better protection against transmission. Testing a panel of antibodies, the authors showed that the antibody S2X324 potently neutralizes all SARS-CoV-2 variants tested, making it a candidate for therapeutic development. A cryo–electron microscopy structure shows how this antibody accommodates Omicron-specific mutations to block binding of the viral spike protein to the human ACE2 receptor across the variants. —VV Vaccine boosters or breakthrough infections elicit plasma-neutralizing activity against Omicron variants.

T he emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant at the end of 2021 caused a worldwide surge in COVID-19 cases. The Omicron BA.1 and BA.1.1 lineages swept the world first, followed by the BA.2 lineage (1). Although BA.1 and BA.2 share a large number of spike (S) mutations, they are each characterized by unique sets of amino acid changes that are associated with different antigenic properties (2)(3)(4). The BA.2.12.1 sublineage emerged in the United States, peaking at the beginning of June 2022, and is characterized by the presence of the L452Q receptor-binding domain (RBD) and S704L fusion machinery mutations in addition to the BA.2-defining mutations (4). The BA.2.75 sublineage is spreading in multiple countries and carries unique mutations (added to the BA.2 background) in the N-terminal domain (NTD), along with D339H, G446S, and N460K mutations and an R493Q reversion in the RBD (5). The BA.3 S glycoprotein comprises a combination of mutations found in BA.1 S and BA.2 S (6), whereas BA.4 S and BA.5 S are identical to each other and comprise a deletion of residues 69 to 70, L452R and F486V substitutions, and an R493Q reversion compared with BA.2 S (7). We characterized the emergence of Omicron (BA.1) as a major antigenic shift because of the unprecedented magnitude of immune evasion associated with this variant of concern (3, [8][9][10][11][12]. Mutations in the BA.1 S glycoprotein NTD and RBD, which are the main targets of neutralizing antibodies (3,8,(13)(14)(15)(16)(17)(18), explain the markedly reduced plasma-neutralizing activity of previously infected or vaccinated subjects (especially those who have not received booster doses) and the escape from most monoclonal antibodies (mAbs) used in the clinic. As a result, an increasing number of reinfections or breakthrough infections are occurring (19)(20)(21)(22), even though these cases tend to be milder than infections of immunologically naive individuals.

Characterization of plasma and mucosal humoral responses to Omicron infection
Understanding the relationships between prior antigen exposure through vaccination or infection with one SARS-CoV-2 strain and the immune response to subsequent infections with a different strain is paramount to guiding strategies to end the COVID-19 pandemic. To investigate this, we first evaluated the magnitude of immune evasion associated with the Omicron sublineages by assessing the neutralizing activity of human plasma using a nonreplicative vesicular stomatitis virus (VSV) pseudotyped with Wuhan-Hu-1 S harboring G614 (Wu-G614), Delta, BA.1, BA.2, BA.2.12.1, or BA.4/5 mutations or with SARS-CoV S ( Fig. 1A;  fig. S1, A to G; table S1; and data S1). We compared plasma from six cohorts of individuals: those previously infected in 2020 (with a Washington-1-like SARS-CoV-2 strain) and then vaccinated twice ("infected-vaccinated 2 doses") or three times ("infected-vaccinated 3 doses); those who were vaccinated and then experienced either a Delta or an Omicron BA.1 breakthrough infection ("Delta breakthrough 3 doses," "BA.1 breakthrough 2 doses," and "BA.1 breakthrough 3 doses"); and those who had only been vaccinated and boosted ("vaccinatedonly 3 doses"). Neutralizing antibody responses were slightly more robust against BA.2 S VSV than against BA.1 S VSV among all groups except for the BA.1 breakthrough cases. Reductions of geometric mean titers (GMTs) relative to Wu-G614 S VSV ranged from 1.4-to 8.2-fold against BA.1 and from 1.6-to 4-fold against BA.2 ( Fig. 1A; fig. S1, A to G; table S1; and data S1), which is consistent with recent findings (4). BA.2.12.1 S VSV was associated with further reductions of plasma-neutralizing activity relative to BA.2 S VSV, whereas BA.4/5 S VSV had the greatest impact of all of the SARS-CoV-2 variants evaluated here, with GMT reductions of 5-to 14-fold relative to Wu-G614 S VSV ( Fig. 1A; fig. S1, A to G; table S1; and data S1). All six cohorts experienced reductions in plasma-neutralizing GMT of 1.4-to 3.6-fold against Delta (23)(24)(25) relative to Wu-G614 S VSV, underscoring that even hybrid immunity [i.e., that acquired through vaccination and infection (26)] does not overcome evasion from neutralizing antibody responses of this previously dominant variant of concern (Fig. 1A; fig. S1, A to G; table S1; and data S1). The highest levels of neutralizing GMTs against SARS-CoV-2 variants were observed for BA.1 breakthrough cases, which was possibly due to exposure to BA.1 S because no correlation was found between time intervals and GMTs (data S1). Neutralizing GMTs against the SARS-CoV S pseudovirus was reduced for all cohorts by 8.6-to 25-fold relative to Wu-G614 S VSV, underscoring the marked genetic and antigenic divergence of this sarbecovirus clade (19,27,28).
Given the recall of Wuhan-Hu-1 plasmaneutralizing antibodies in Omicron breakthrough cases, we investigated the cross-reactivity of RBD-directed antibodies produced by in vitrostimulated memory B cells obtained up to 200 days after infection or vaccination, as well as in circulating plasma cells collected in the days after infection (29). These analyses used blood samples from individuals who were infected before the emergence of Omicron and subsequently vaccinated ("infected-vaccinated 2/3 doses"), as well as subjects who experienced either an Omicron primary infection or an Omicron breakthrough infection. Primary and breakthrough Omicron infections occurred between January and March 2022, during which time the prevalence of Omicron BA.1/BA.2 sublineages exceeded 90% in the region from which the samples were obtained ( fig. S2). Plasmaneutralizing activity of Omicron-infected (primary and breakthrough) cases was reduced an average of 6.1-fold against BA.4/BA.5 S VSV relative to BA.1 S VSV (table S2), likely as a result of both RBD and NTD mutations in the former lineage, concurring with the above data and recent studies (30,31). More than 80% of SARS-CoV-2 RBD-directed IgGs secreted by memory B cells and plasma cells obtained from breakthrough cases cross-reacted with the Wuhan-Hu  S4, D to F). This suggests that the maturation of antibodies driven by BA.1 or BA.2 breakthrough infections may also result in a narrowing of their specificity over time, thereby decreasing crossreactivity with the BA.4/5 RBD. These findings illustrate how immunological imprinting from prior exposure, also referred to as "original antigenic sin," can strongly affect the response to distantly related antigens. By contrast, memory B cell-derived RBD-directed IgG antibodies obtained from Omicron primary infections up to 6 to 7 months after infection were present at low frequency and were mostly specific for the BA.1 and BA.2 RBDs, (Fig. 1B, figs. S3 to S6, and data S1). The frequency of IgG antibodies crossreacting with the SARS-CoV RBD was similar across all three cohorts, concurring with the overall weak plasma-neutralizing activity (Fig.  1, A and B, and table S2).
We determined the site specificity of RBDdirected antibodies secreted by stimulated memory B cells by competition with structurally characterized mAbs targeting four distinct antigenic sites (13,27). Most of the memory B cell-derived antibodies from (pre-Omicron) infected-vaccinated individuals competed with the five reference mAbs used, whereas a large fraction of antibodies from Omicron break-through cases did not compete with any of these five mAbs, indicating that they recognize other undefined RBD antigenic sites ( Fig.  1C and fig. S7). Antibodies recognizing most antigenic sites overlapping with the receptorbinding motif (RBM), such as mAb S2E12 (33), were found at lower frequency upon Omicron breakthrough infections relative to infectedvaccinated subjects, consistent with the presence of several immune escape mutations in the Omicron RBM ( Fig. 1C and fig. S7) (3,18). A similar relative reduction was observed for antibodies targeting RBD antigenic site IIa [recognized by the S2X259 mAb (34)] ( Fig. 1C  and fig. S7), in agreement with previous findings describing Omicron immune escape from several site IIa mAbs (3,8,18). Collectively, these findings demonstrate that Omicron breakthrough infections preferentially expand existing B cell pools primed by vaccination and elicit crossreactive antibodies, supporting the concept of immunological imprinting.
To evaluate mucosal antibody responses in subjects who experienced a BA.1 breakthrough infection or in vaccinated-only subjects, we assessed IgG-and IgA-binding titers in nasal swabs obtained longitudinally after polymerase chain reaction (PCR) testing. Although we detected S-specific IgG, and to a lesser extent IgA, in swabs from several breakthrough cases, ; and data S1). Furthermore, analysis of nasal swabs obtained from four breakthrough cases~6 months after symptom onset demonstrated a retention of neutralizing activity. , and SARS-CoV S VSV pseudoviruses using plasma from subjects who were infected and vaccinated, vaccinated and experienced breakthrough infection, or received vaccination only. VeroE6-TMPRSS2 cells were used as target cells (93). Data are the geometric mean of an n = 2 technical replicates and have been performed in at least two biologically independent experiments. GMTs are shown with a color-matched bar (and reported in table S1) with the fold change compared with Wu-G614 indicated above. Demographics of enrolled donors are provided in data S1. (B) Cross-reactivity of IgGs secreted from memory B cells obtained from infected-vaccinated individuals (n = 11), primary SARS-CoV-2 infected individuals (n = 3 samples collected at 1 to 3 months and n = 2 samples collected at 6 to 7 months), or breakthrough cases (n = 7 samples collected at 1 to 3 months and n = 4 samples collected at 4 to 6 months) occurring in January-March 2022, when the prevalence of Omicron BA.1/BA.2 exceeded 90% in the region where samples were obtained ( fig. S2). Each dot represents a well containing oligoclonal B cell supernatant screened for the presence of IgGs binding to the SARS-CoV-2 Wuhan-Hu-1 and BA.1 RBDs (top) or to the SARS-CoV-2 Wuhan-Hu-1 and SARS-CoV RBDs (bottom) using ELISA. Red dots indicate inhibition of the interaction with ACE2 (using Wuhan-Hu-1 target antigen) as determined in a separate assay. The percentages are expressed relative to the total positive hits against any of the antigens tested. Numbers of positive hits relative to individual donors are shown in fig. S3. (C) Frequency analysis of site-specific IgG antibodies derived from memory B cells. RBD sites targeted by IgG derived from memory B cells were defined by a blockade-of-binding assay using mAbs specific for sites Ia (S2E12), Ib (S2X324), IIa (S2X259), IV (S309; parent of sotrovimab), and V (S2H97). Hybrid sites Ia/Ib, Ia/IIa, Ib/IIa, Ib/IV, IIa/V, and IV/V were defined by competition with the two corresponding mAbs. Hybrid sites exhibiting competition with more than two mAbs are indicated as "multiple. " Lack of competition is indicated as "other." Pie charts show cumulative frequencies of IgGs specific for the different sites among total RBD-directed IgG antibodies (left) and those inhibiting binding of RBD to human ACE2 (right) in n = 11 infected-vaccinated individuals or n = 7 breakthrough cases. Assessing plasma-neutralizing antibody titers of these BA.1 breakthrough cases yielded similar magnitude and GMT reductions compared with the rest of the BA.1 breakthrough cohort (Fig. 1A, fig. S1F, and data S1). The magnitude of the neutralizing antibody responses in nasal swabs cannot be directly compared with plasma samples because of the self-administration procedure and resulting sample nonuniformity. Overall, we observed heterogeneous mucosal neutralizing antibody responses among BA.1 breakthrough cases but not in vaccinatedonly individuals (Fig. 1, D and E; fig. S9, C and D; and data S1). Collectively, these data underscore the lack of or very weak induction of mucosal antibody responses upon intramuscular delivery of mRNA vaccines or adenovirusvectored vaccines (35,36) and are consistent with concurrent findings that Omicron breakthrough infection, but not vaccination alone, induces neutralizing antibody responses and tissue-resident T cells in the nasal mucosa (37,38).

Omicron sublineages escape neutralization mediated by most clinical mAbs
We next evaluated the impact of BA.1, BA.  (18). The importance of this site was also identified through deep mutational scanning (39), and this point mutation was shown to reduce neutralizing activity by~4-fold for COV2-2130 (8). The greater reduction in potency against BA.4 and BA.5 relative to BA.2 is likely driven by the L452R mutation, as reported (https://www. fda.gov/media/154701/download) (39). The REGN10987 + REGN10933 and LY-CoV16 + LY-CoV555 mAb cocktails and the CT-P59 and ADI-58125 mAbs had reductions of in vitro neutralization potency ranging between two and four orders of magnitude against all Omicron sublineage S VSV pseudoviruses compared with Wu-D614 S VSV because of mutations in the RBM ( Fig. 2A and fig. S10, A and B) (18). CT-P59, however, retained neutralizing activity against the BA.2.75 sublineage (29.2-fold reduction relative to Wu-D614 S VSV). The recently described ACE2-mimicking S2K146 mAb (40), which retained unaltered activity against BA.1 compared with Wu-D614 (3), had a mildly reduced neutralizing activity against BA.2, BA.3, BA.2.12.1, and BA.2.75 S VSV pseudoviruses (3.3-, 3.1-, 1.9-, and 4.3-fold, respectively) ( Fig. 2A and fig. S10, A and B) Fig. 2A). Similar reductions in neutralizing activity were also observed against authentic Omicron sub-lineage virus isolates (Fig. 2C and fig. S11), and are greater than that observed against BA.1 pseudovirus (2.7-fold), although no additional residue mutations map to the sotrovimab epitope except the G339H substitution present in BA.2.75 instead of G339D found in BA.1 (41)(42)(43). We recently showed that sotrovimab retained in vitro effector functions against BA.2 and conferred Fc-dependent protection in the lungs of mice infected with BA.2 (44). The additional loss of neutralization of these Omicron sublineage VSV pseudoviruses beyond BA.1 likely results from the S371F substitution, which is found in BA.2, BA.3, BA.4/5, BA.2.12.1, and BA.2.75, and introduces a bulky phenylalanine near the N343 glycan, which is part of the sotrovimab epitope (41). A recently determined BA.2 S structure shows that the RBD helix comprising residues 364 to 372 is indeed remodeled (45) and adopts a distinct conformation from the ones observed for Wuhan-Hu-1 S or BA.1 S structures (18,46). This structural rearrangement is sterically incompatible with the glycan N343 conformation observed in S309-bound spike structures (18,41), as supported by molecular dynamics simulations, and likely explains the reductions in neutralization potency ( fig. S11, A to D Fig. 2A and fig. S10, A and B), likely because of the detrimental effect of the aforementioned S371F/S373P/S375F-induced remodeling and of the R408S mutation (34). S2H97 is a site V mAb that had a 4.7-to 10-fold decrease in neutralization potency against Omicron sublineages compared with Wu-D614 S VSV ( Fig. 2A and fig. S10, A and B) despite the absence of mutations present in the epitope or otherwise found to affect binding by DMS, perhaps reflecting differential accessibility to its cryptic epitope in the context of these S trimers (27).
To evaluate the ability of S2X324 to promote antibody dependent-phagocytosis or cytotoxicity, we tested whether the mAb could activate Fcγ receptors expressed at the surface of Jurkat cells. Although S2X324 only activated FcγRIIIa, but not FcγRIIa, in vitro ( fig. S15, A and B), it triggered both antibody-dependent phagocytosis and cytotoxicity after incubation of     peripheral blood mononuclear cells with SARS-CoV-2 S-expressing cells ( fig. S15, C to F). The slow S 1 shedding kinetics likely explain the ability of S2X324 to promote Fc-mediated effector functions.

Structural basis for S2X324-mediated neutralization
To understand the pan-variant S2X324 inhibitory activity, we determined a cryo-electron microscopy structure of the Omicron BA.1 S ectodomain trimer bound to the S2X324 Fab fragment at 3.1-Å resolution (Fig. 3A, fig. S16, and table S5). In our structure, the BA.1 S trimer had three Fabs bound to one closed and two open RBDs. We used focused classification and local refinement of the closed RBD-S2X324 Fab complex to obtain a 3.3-Å structure revealing the molecular details of the binding interface.
The structure explains how this mAb accommodates residues that are mutated in Omicron lineages relative to Wuhan-Hu-1: N440K (BA. 1 fig. S13A) might be caused by G446S, because although the mutation is clearly accommodated, at least one of three favored rotamers for S446 would clash with the Fab. The Y501 backbone forms van der Waals interactions with the VL N32 side chain that are independent of the RBD residue identity at position 501 (explaining retention of neutralization of all Y501-containing variants). S2X324 and LY-CoV1404 share 87 and 91% amino acid sequence identity in their heavy and light chains, respectively, likely explaining their similar binding mode ( fig. S17) (48), pan-variant neutralizing activity (49), and comparable resilience to Omicron sublineage mutations thus far ( Fig. 2A).

Identification of S2X324 viral escape mutants in vitro
To explore potential mutations that could promote escape from S2X324-mediated neutralization, we passaged a replication-competent VSV chimera harboring either SARS-CoV-2 Wu-G614 S (50) or Omicron BA.1 S in the presence of S2X324. Residue substitutions at three distinct sites emerged in both S backgrounds ( Fig. 3C; fig. S18 1 background), which might alter the local RBD backbone conformation and/or sterically hinder mAb binding. Furthermore, three additional mutations were detected in the BA.1 S background only, S446I, G447S, and N448K, which are positioned near the interface between the heavy and light chains ( Fig. 3C; fig. S18, A and B; and tables S6 and S7). The VSV chimera harboring SARS-CoV-2 Wu-G614 S outcompeted the chimeras harboring the K444T/N, V445D, or P499R escape mutants after four rounds of passaging, suggesting reduced fitness in this replicating chimeric virus model system ( fig. S18C). Even though each of these mutations requires a single nucleotide substitution, they are very rare and have been detected cumulatively only in 0.087 and 0.080% of Delta and Omicron genome sequences as of 12 August 2022, respectively (table S8 and fig. S19), although the frequency of some of them is increasing. We further tested VSV pseudoviruses bearing Wu-G614, BA.1, or BA.2 S carrying K444E, K444D, K444N, K444T, V445D, and P449R/H, and confirmed that these mutations abrogated or strongly reduced S2X324-neutralizing activity ( fig. S19 and  table S9). In addition, S2X324-neutralizing activity was abrogated when V445T/A/F was introduced in the BA.1 backbone (table S9). S2X324 retained potent neutralizing activity 6 of 9 against pseudoviruses bearing other mutations in the epitope found in known variants such as N439K, N440K, and N501Y in the Wu-G614 S background (table S9). Although the S2X324 escape mutants identified are rare, these data suggest that a mAb cocktail comprising S2X324 would increase the barrier for the emergence of resistance mutants even further compared with this single mAb.
S2X324 protects hamsters against SARS-CoV-2 Delta, BA.2, and BA.5 variants We investigated the in vivo prophylactic and therapeutic efficacy of S2X324 using Syrian hamsters challenged with SARS-CoV-2 variants. Prophylactic administration of S2X324 or S309 comparably protected hamsters chal-lenged with SARS-CoV-2 Delta in a dosedependent manner (Fig. 4, A to C) despite a 20-fold difference in in vitro potency against SARS-CoV-2 Delta S VSV (Fig. 2B). These data support the lack of direct correlation between in vitro and in vivo potency that was previously reported (51,52). Moreover, prophylactic administration of S2X324 at 5 mg/kg decreased viral loads below detection levels in the lungs of hamsters challenged with BA.2 or BA.5 (Fig.  4, D to F). In this model, S309 retained activity against BA.5 despite a 22.6-fold reduced in vitro potency relative to Wu-D614 (Fig. 2, A and B). Therapeutic administration of hamster IgG2a S2X324 (1 day after challenge with the SARS-CoV-2 Delta variant) at 2 and 5 mg/kg prevented body weight loss and reduced lung viral RNA loads by 2.5 and 3 orders of magnitude compared with the control group, respectively (Fig. 4, G and H). Viral replication in the lungs was fully abrogated at 2 and 5 mg/kg of S2X324 and reduced by about one order of magnitude for animals treated with 0.1 and 0.5 mg/kg of S2X324 (Fig. 4I). No statistically significant differences were observed for animals receiving an Fc-silenced version of S2X324 (N297A) versus the groups receiving the same doses of Fc-competent S2X324, indicating that limited contribution of Fc-mediated effector functions in these experimental conditions.

Discussion
Immune imprinting, which is also referred to as original antigenic sin, was described based Park   , and viral RNA load (C). n = 6 animals/dose. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 relative to isotype control (MGH2 mAb against circumsporozoite protein of Plasmodium sporozoites). Data were analyzed with Kruskal-Wallis test followed by Dunn's multiple-comparisons test. (D) Quantification of viral RNA loads in the lung and trachea of Syrian hamsters 4 days after intranasal infection with SARS-CoV-2 Omicron BA.2, which was preceded 1 day prior by prophylactic intraperitoneal administration of S2X324 hamster IgG2a at 5 mg/kg of body weight. ***P < 0.001 relative to control. Data were analyzed with Mann-Whitney two-tailed t test. (E and F) Quantification of replicating virus titers (TCID 50 ) (E) and viral RNA load (F) in the lung of Syrian hamsters 4 days after intranasal infection with SARS-CoV-2 Omicron BA.5, which was preceded 1 day prior by prophylactic intraperitoneal administration of S309 or S2X324 human IgG1 (HuS309 and HuS2X324). (G to I) Dose-dependent protection in animals 4 days after infection with SARS-CoV-2 Delta by therapeutic intraperitoneal administration of S2X324 hamster IgG2a (blue symbols) or the S2X324 N297A mutant IgG2a (purple symbols) 1 day later at 5, 2, 0.5, or 0.1 mg/kg of body weight. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 relative to control, respectively. Data were analyzed with Mann-Whitney two-tailed t test.
on the observation that infections with influenza virus strains distinct from the one that caused prior infection preferentially boosted antibody responses against epitopes shared with the original strain (53). Although this phenomenon is often considered detrimental, it can also be beneficial, as was the case at the time of the 2009 H1N1 pandemic, during which initial antibody responses to infection with this newly emerged and antigenically shifted virus were dominated by antibodies targeting the conserved hemagglutinin stem region (54,55). Subsequent exposures through vaccination or infection elicited antibody responses to the shifted variant (i.e., to "nonconserved" hemagglutinin epitopes) (54,56). Moreover, several studies reported hemagglutinin stem-directed antibody-mediated protection against H5N1 and H7N9 zoonotic influenza strains through imprinting during childhood resulting from exposure to seasonal H1N1 and H3N2, respectively (55,57). Similarly, we show that exposure to antigenically shifted Omicron strains primarily recalls existing memory B cells specific for epitopes shared by multiple SARS-CoV-2 variants rather than priming naïve B cells recognizing Omicron-specific epitopes (at least up to 180 days after breakthrough infection), as was also recently reported (58). Although immune imprinting may be beneficial for stimulating responses to cross-reactive SARS-CoV-2 S epitopes, antibody responses to some Omicron S-specific epitopes were hindered by prior antigenic exposure.
Currently, there is uncertainty whether vaccines matching dominant circulating SARS-CoV-2 variants such as those used for seasonal influenza are needed, or if the repeated use of Wuhan-Hu-1-based vaccines will suffice. Recent work showed that boosting previously immunized macaques with Beta or Omicron mRNA S vaccines or with Beta RBD nanoparticle vaccines elicited comparably high titers of antibodies broadly neutralizing multiple variants relative to Wuhan-Hu-1-based vaccines (59)(60)(61). Furthermore, administration of Wuhan-Hu-1-based vaccine boosters in humans was shown to elicit appreciable titers of neutralizing antibodies and prevent severe disease associated with Omicron infections (11,19,(62)(63)(64)(65). The limited cross-variant neutralization elicited by Omicron primary infection in humans or Omicron-based vaccination of immunologically naïve animals and the data on the specificity of memory B cells presented here indicate that an Omicron-based vaccine might elicit antibody responses directed toward the vaccinematched and closely related antigens. This suggests that a heterologous prime boost or a multivalent approach might be preferable (59,(66)(67)(68)(69)(70)(71)(72)(73). Omicron infection and Omicron S-based vaccination of previously immune subjects, however, recalls cross-reactive memory B cells (58,74), which may further mature over time to enhance their affinity and neutralizing potency against Omicron, but also to possibly broaden their neutralizing activity against past and future variants. Indeed, multiple studies have shown that somatic hypermutations yield RBD-specific mAbs with increased affinity for the homotypic antigen and augmented resilience to immune evasion of emerging heterotypic variants (40,(75)(76)(77)(78)(79). The recently introduced bivalent mRNA vaccine boosters encoding the Wuhan-Hu-1 and either the BA.1 or the BA.4/5 S glycoproteins have yielded encouraging results (80)(81)(82).
Understanding antibody responses elicited by and directed toward Omicron sublineages is as the result of key to informing public health policies and the design of SARS-CoV-2 and sarbecovirus vaccines (70,71,(83)(84)(85). Our data show that Omicron breakthrough infections do not elicit high titers of pan-sarbecovirus-neutralizing antibodies (e.g., directed against SARS-CoV), in agreement with recent data (86). These findings contrast with the observation that preexisting immunity to SARS-CoV followed by SARS-CoV-2 vaccination is associated with elicitation of pan-sarbecovirusneutralizing antibodies (28). These different outcomes might be explained by the low frequency of memory B cells encoding neutralizing antibodies targeting antigenic sites shared among pre-Omicron variants (Wuhan-Hu-1related strains), Omicron, and SARS-CoV because of the genetic and antigenic distances between these three distinct viruses. For instance, Omicron BA.1 and BA.2 harbor variations of the RBD antigenic site II, which is the target of pan-sarbecovirus-neutralizing antibodies such as S2X259 (34), DH1047 (87), and ADG2 (88), leading to resistance to the neutralization mediated by some of these mAbs (3,8,18). This suggests that conservation of RBD antigenic sites across sarbecoviruses may have resulted (at least partially) from limited immune pressure rather than from functional or structural constraints (i.e., some mutations at these conserved sites may remain compatible with viral fitness) (86).
Recent preclinical assessment of intranasally administered influenza and sarbecovirus vaccine candidates has demonstrated the induction of lung-resident protective mucosal humoral and cellular immunity at the site of viral entry (89)(90)(91)(92). These observations, along with our findings that SARS-CoV-2 breakthrough infections, but not vaccination alone, elicit neutralizing activity in the nasal mucosa, support the development and evaluation of a next generation of vaccines administered intranasally.