Sequence Matters: Primary COVID-19 Vaccination after Infection Elicits Similar Anti-spike Antibody Levels, but Stronger Antibody Dependent Cell-mediated Cytotoxicity than Breakthrough Infection

Since the zoonotic introduction of SARS-CoV-2 in 2019, population-based immunity has steadily risen through waves of infection with rapidly evolving variants and through mass immunization programs with less rapidly evolving vaccines (1–3). Studies of COVID-19 vaccine efficacy found that appropriate intervals between immunizations are important to maximize boosting of immunity from either previous infection or vaccination (4, 5). To optimize immune responses and maximize vaccine distribution, Canadian guidelines currently recommend a 6-mo interval between infection or vaccination and subsequent vaccine boosting (6). High-throughput evaluation of vaccine efficacy is primarily based on levels of IgG induced against SARS-2-CoV spike (S) protein and associated neutralization capacity. Neutralization of the SARS-CoV-2 variant represented in vaccines or in previous infection correlates well with the total level of anti-S Ab and more closely with the level of Ab against the receptor binding domain (RBD) within the S protein of the variant under consideration (7–10). Mutations within the RBD reduce neutralization capacity against SARS-CoV-2 variants and enable breakthrough infections, but the reduced severity of illness with breakthrough infection implies that the antiviral activity of immune processes other than neutralization are more conserved and can contribute to a greater breadth of protection (1, 3, 11–20).

Following the initial waves of infection with antecedent strains of SARS-CoV-2, evidence emerged that hybrid immunity induced by a single vaccination in those previously infected with SARS-CoV-2 was more robust than anti-SARS-CoV-2 immunity induced by two or even three vaccinations in those never infected (12, 13, 19, 21, 22). The severity of COVID-19 has generally lessened in parallel with the evolution of SARS-CoV-2 into the currently dominant Omicron family of variants (23). To what extent this reflects the higher background of population-based immunity versus an intrinsic decrease in viral virulence is not completely clear, but studies clearly indicate that the strength of immunity postinfection reflects the severity of infection (21, 24–26). In addition, immune imprinting from primary antigenic exposure is a relevant factor affecting the fine specificity and functionality of humoral immunity boosted by secondary exposure (27–29). Therefore, it is important in these contexts to investigate how breakthrough infection with newer, less virulent SARS-CoV-2 variants modulates the strength and breadth of humoral immunity elicited by vaccination. To address this question, we assessed aspects of humoral immunity against SARS-CoV-2 Wuhan-Hu-1 and Omicron (BA.1) S following three Wuhan-Hu-1 S-based mRNA vaccinations and again after Omicron breakthrough infection. Our previous study demonstrated that hybrid immunity elicited a more robust capacity for Ab-dependent cell-mediated cytotoxicity (ADCC) than vaccination, despite equivalent numbers of antigenic exposures and stimuli for affinity maturation (30). This raised the question whether infection after vaccination would shift the potential for robust ADCC in a group of participants who had previously been exposed only through vaccination. We measured total anti-S and anti-RBD IgG and IgA levels, IgG reactivity against S1 and S2, and the capacity to elicit ADCC before and after Omicron breakthrough infection and compared these levels with hybrid immunity to determine whether differences in immunization sequence or type have significant quantitative and qualitative effects on humoral immunity.

This study complies with the recommendations of the Canadian Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans, and ethical approval was given by the Health Research Ethics Authority of Newfoundland and Labrador. Peripheral blood was collected from study subjects at approximately 3-mo intervals, and a questionnaire addressing previous testing history and reasons for suspecting infection with SARS-CoV-2 was administered at study intake after written informed consent in accordance with the Declaration of Helsinki.

Eighteen individuals receiving three doses of a COVID-19 mRNA vaccine with no previous COVID-19 infection history before Omicron variant breakthrough infection were matched with 31 previously identified individuals with confirmed Wuhan-Hu-1 infection prior to receiving their first COVID-19 vaccine (Table I) (30, 31). Participants self-declared any medical treatments they were receiving as well as information on comorbidities. Persons with known immune-compromising conditions or receiving immunosuppressive treatment were excluded from this cohort.

Whole blood was collected by venipuncture into acid citrate dextrose vacutainers, and PBMCs and plasma were fractionated by density gradient centrifugation as per the Canadian Autoimmunity Standardization Core consensus standard operating procedure (version: March 21, 2019). Plasma was stored at −80°C until use. For ADCC experiments, PBMCs were isolated from acid citrate dextrose anticoagulated whole blood from healthy donors as above, resuspended in complete lymphocyte medium consisting of RPMI 1640 with 10% FBS (HyClone), 200 IU/ml penicillin/streptomycin, 0.01 M HEPES, 1% l-glutamine (all from Invitrogen, Carlsbad, CA), and 2 × 10⁻⁵ M 2-ME (Sigma-Aldrich) and used directly in functional experiments.

Cell lines and PBMCs were cultured with 5% CO₂ at 37°C. Human lung fibroblast MRC-5 cells were obtained from ATCC (CCL-171) and propagated in complete DMEM (Sigma-Aldrich) containing 10% FBS (HyClone) and 200 IU/ml penicillin/streptomycin (Invitrogen). MRC-5 cells stably expressing Wuhan-Hu-1 or Omicron (BA.1) S Ag were previously constructed using the Lenti-X pLVX-IRES lentiviral vector expression system (TakaRa) (21, 30). Transduced cells were selected in complete DMEM containing 1 μg/ml puromycin (Sigma-Aldrich). Extracellular S expression was confirmed by flow cytometry, as previously outlined (21).

Freshly isolated PBMCs from three healthy donors were used in ADCC assays as described previously (21, 30). In brief, after confirmation of equivalent levels of S expression, 10⁴ Wuhan-Hu-1 or Omicron S-expressing MRC-5 target cells were plated per well and labeled with 1 μCi Na₂⁵¹CrO₄/well (PerkinElmer, Akron, OH) overnight in 96-well round-bottomed plates at 37°C with 5% CO₂. After washing, heat-inactivated plasma (56°C for 1 h) and PBMCs (E:T 25:1) were added to wells in duplicate with a final volume of 300 μl and final plasma dilution of 1:1000 or 1:250. Cytotoxicity was measured over 5 h by ⁵¹Cr-release in 125 μl of supernatant on a Wallac 1480 Wizard gamma counter, with the percentage specific lysis calculated as follows: (experimental ⁵¹Cr release – spontaneous ⁵¹Cr release)/(maximum ⁵¹Cr release – spontaneous ⁵¹Cr release) × 100. Prepandemic heat-inactivated plasma ADCC controls were included in all assays, and this background signal was subtracted from plotted values.

Serology was performed as previously outlined (21, 30, 31). Briefly, plasma was diluted at 1:250 and 1:1000, and total IgG or IgA reactivity was tested against (1) 50 ng/well SARS-CoV-2 Wuhan-Hu-1 (SMT1-1 reference material, National Research Council Canada [NRC]) or Omicron (B.1.1.529 SmT1v3 reference material; NRC) full-length spike (FLS) glycoprotein trimer, (2) 50 ng/well Wuhan-Hu-1 (SinoBiological, Wayne, PA) or Omicron (ACROBiosystems, Newark, DE) RBD to indirectly address the neutralization capacity of plasma Abs, or (3) plasma was diluted at 1:100 and total IgG reactivity was tested against the S1 subunit of SARS-CoV-2 S (65 ng/well; SinoBiological) and S2 subunit of SARS-CoV-2 S (50 ng/well; SinoBiological). Coating amounts for S1 and S2 were adjusted to account for differences in their predicted molecular mass. Total IgG was measured using 1:50,000 HRP-conjugated polyclonal goat anti-human IgG (Jackson ImmunoResearch Labs, West Grove, PA), total IgA was measured using 1:20,000 HRP-conjugated polyclonal goat anti-human IgA (Jackson ImmunoResearch Labs) and developed using tetramethylbenzidine (TMB) substrate (Sigma-Aldrich). Reactions were stopped after 20 min with an equal volume of 1 M H₂SO₄, and OD was read on a BioTek synergy HT plate reader at 450 nm.

In parallel with the ⁵¹Cr-release assay, 96-well round-bottomed plates were coated with 10⁴ Wuhan-Hu-1 or Omicron S-expressing MRC-5 cells per well overnight at 37°C with 5% CO₂. Culture medium was decanted, plates were washed as in the ⁵¹Cr-release assay (21, 30), and heat-inactivated plasma was applied to wells (100 µl/well) in duplicate at a final dilution of 1:250 and incubated with 5% CO₂ at 37°C for 1.5 h. Diluted plasma was decanted, plates washed three times with PBS containing 1% FBS, then fixed using 50 μl/well of 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature. Nonspecific Ags were blocked for 1 h using 200 μl/well of PBS containing 1% BSA (Sigma-Aldrich). Total IgG was measured using1:50,000 HRP-conjugated polyclonal goat anti-human IgG (Jackson ImmunoResearch Labs) and developed using TMB substrate (Sigma-Aldrich). Reactions were stopped after 20 min with an equal volume of 1 M H₂SO₄, and OD was read on a BioTek synergy HT plate reader at 450 nm.

Individual overlapping peptides (180 17-mers and one N-terminal 13-mer, with 10-aa overlaps) spanning the canonical Wuhan-Hu-1 S sequence (NR-52402; Biodefense and Emerging Infections Research Resources Repository [BEI Resources]) were reconstituted at 10 mg/ml in DMSO (Sigma-Aldrich), then diluted to 50 μg/ml in Dulbecco’s PBS (Sigma-Aldrich) and stored at −20°C as previously described (30). Each S peptide 2–181 (BEI Resources) at 125 ng/well was coated overnight on a 96-well OptiPlate HB (PerkinElmer, Woodbridge, ON, Canada) in sequential pairs (e.g., 2 and 3 … 180 and 181). Peptide 1, representing the signal sequence leader peptide, was coated on its own at 125 ng/well. FLS trimer (SMT1-1, NRC) was diluted in Dulbecco’s PBS and coated overnight at 150 ng/well as a positive control. Plates were washed four times with 0.05% PBST and blocked for 1 h with 200 μl/well 5% nonfat powdered milk (Carnation) in 1× Tris-buffered NaCl (Sigma-Aldrich). Plasma was diluted 1:50 in 0.05% PBST + 1% nonfat powdered milk, and 50 μl was applied for 1.5 h. Plates were washed six times, and total IgG binding was measured in a 1-h incubation with 100 μl/well of 1:50,000 HRP-conjugated polyclonal goat anti-human IgG (Jackson ImmunoResearch Labs) and developed with 50 μl/well of a 1:2 dilution with dH₂O of SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher). Reactions were read within 20 min of developing on a Fluoroskan Ascent FL luminescence reader. Background signal (diluent only) was subtracted from all values, and corrected relative light unit (RLU) values obtained from the hybrid immunity group were normalized to those acquired from the breakthrough infection cohort and plotted.

To allow a more accurate assessment of plasma Ab avidity to Wuhan-Hu-1 and Omicron FLS, we first serially diluted participant plasma using the standard serological ELISA outlined above to titrate each plasma sample to determine the optimal dilution that fits the linear part of the titration curve, designated as OD 1, for each participant’s plasma sample (32). Plasma samples were subsequently tested for avidity by ELISA at the plasma dilution corresponding to OD 1 for 50 ng/well of recombinant Wuhan-Hu-1 (SMT 1-1, NRC) or Omicron B.1.1.529 (SmT1v3, NRC) FLS protein coated overnight onto 96-well Immulon-2 plates (VWR Scientific). Plates were washed four times, blocked for 1 h, washed four times, and then 100 μl/well of diluted plasma was applied to Ag-coated plates in quadruplicate wells for 1.5 h. Plates were washed two times, 100 μl/well of 8 M urea (Sigma-Aldrich) or PBS was applied in duplicate for 10 min, followed by four washes and a 1-h incubation with 100 μl/well of 1:50,000 HRP-conjugated polyclonal goat anti-human IgG (Jackson ImmunoResearch Labs). Plates were developed using TMB substrate (Sigma-Aldrich) following six washes, then incubated in the dark at room temperature for 20 min. Reactions were stopped with an equal volume of 1 M H₂SO₄, and OD was read on a BioTek synergy HT plate reader at 450 nm. To calculate Ab binding (avidity index) to Wuhan-Hu-1 or Omicron FLS protein, the mean OD (450 nm) value of the sample subjected to urea (chaotropic reagent) treatment was divided by the value of the sample without urea treatment (PBS) and expressed as a percentage.

Statistical analyses were performed using GraphPad Prism 9, with two-sided p values <0.05 considered significant. Normality of data distributions were assessed using Kolmogorov–Smirnov test. Differences in means with SDs or medians with interquartile ranges (IQRs; calculated as IQR = Q₃ − Q₁) between groups were compared by using one-way ANOVA, Kruskal–Wallis test, Friedman test, Student t test or Mann–Whitney U test as appropriate on the basis of the normality of data distribution.

To determine whether SARS-CoV-2 infection itself or the sequence by which we are exposed through vaccination and infection underlies the generation of Ab eliciting potent ADCC, we compared ADCC elicited by plasma IgG from two cohorts (Table I) against MRC-5 cells expressing SARS-CoV-2 S protein. This established platform was used previously to selectively focus on S-specific ADCC and was validated in parallel with SARS-CoV-2–infected cells (30). At a 1:1000 plasma dilution, ADCC elicited against MRC-5 cells expressing Wuhan-Hu-1 S with plasma from participants who had received three vaccines and from the same participants following Omicron breakthrough infection was significantly weaker (p < 0.0001) than that elicited by plasma from the hybrid immunity group that had received one vaccination following Wuhan-Hu-1 infection (Fig. 1A). Despite two additional antigenic exposures, this Fc-dependent function of IgG Ab against SARS-CoV-2 was lower in the breakthrough infection group. Omicron breakthrough infection did not increase the capacity to elicit ADCC at the 1:1000 plasma dilution tested (Fig. 1A). To investigate reasons for this, we first tested plasma samples from the two groups to (1) assess the impact of Omicron breakthrough infection and (2) compare relative levels of IgG Ab against plate-bound Wuhan-Hu-1 FLS and RBD induced by Wuhan-Hu-1 infection before vaccination to levels induced by Omicron infection following three vaccinations. Previous infection prior to Omicron breakthrough was ruled out by serial testing for anti-SARS-CoV-2 nucleoprotein (N) Ab (31). At a 1:1000 dilution of plasma, Omicron breakthrough infection produced a significant increase in IgG against plate-bound Wuhan-Hu-1 FLS (p = 0.0299; Fig. 1B) and RBD (p = 0.0232; Fig. 1C) compared with levels after three vaccinations.

Despite similar total IgG levels against FLS and RBD after Omicron breakthrough infection and in the hybrid immune cohort (Fig. 1B, 1C), ADCC elicited against MRC-5 cells expressing SARS-CoV-2 S proteins using a 1:1000 plasma dilution was significantly less for the Omicron breakthrough infection cohort (Fig. 1A). A plasma dilution of 1:250 for the Omicron breakthrough cohort produced ADCC levels similar to those obtained with plasma diluted 1:1000 from subjects vaccinated following Wuhan-Hu-1 infection, and this 1:250 dilution was used for further studies. Despite the significant increase in IgG anti-FLS levels at the higher 1:1000 plasma dilution (Fig. 1B), Omicron breakthrough infection did not significantly increase ADCC elicited against either Wuhan-Hu-1 or Omicron S-expressing MRC-5 cells, and preferred recognition of Wuhan-Hu-1 S through ADCC persisted after Omicron breakthrough (Fig. 2A). Expression of Wuhan-Hu-1 and Omicron S was maintained at equivalent levels on MRC-5 target cells for the assay (Fig. 2B). Despite the breakthrough infection cohort having overall anti-S IgG levels similar to the hybrid immunity cohort (Fig. 1B), a 4-fold higher plasma concentration (1:250 versus 1:1000) was required to elicit comparable levels of ADCC (Fig. 2A). Thus, features beyond overall level of IgG against SARS-CoV-2 S influence the capacity of the humoral immune response generated by vaccination and infection to elicit robust ADCC against SARS-CoV-2 S-expressing targets.

Direct comparison of Ab levels at the 1:250 plasma dilution used to elicit ADCC indicated no significant difference between plasma IgG levels induced against Wuhan-Hu-1 or Omicron FLS by three vaccinations and levels following Omicron breakthrough infection (Fig. 2C). However, consistent with the increased neutralization capacity reported following Omicron breakthrough infection (33–35), there was a significant increase in plasma IgG against Wuhan-Hu-1 and Omicron RBD (Fig. 2D) to levels similar to those seen in the hybrid immunity group (Fig. 1C and data not shown). Preferred recognition by plasma IgG of Wuhan-Hu-1 S over Omicron S persisted after Omicron infection (Fig. 2C–2E), illustrating the phenomenon of humoral imprinting from initial and repeated exposure to Wuhan-Hu-1 S and its impact on functional responses (Fig. 2A). There was a significant increase (p < 0.0001) in IgG binding to S expressed in situ on the MRC-5 cell surface selective for Omicron (Fig. 2E), but despite this selective increase, preferred recognition of cell surface Wuhan-Hu-1 S was maintained, and ADCC was not significantly increased against Wuhan-Hu-1 or Omicron-S expressing targets (Fig. 2A).

Although circulating IgA Abs do not support ADCC (36–39), they can neutralize SARS-CoV-2 and potentially interfere with ADCC by competing with IgG Abs for relevant binding sites. Therefore, we measured and compared plasma levels of IgA Abs against Wuhan-Hu-1 and Omicron FLS and RBD and tested for correlation with ADCC capacity. Although IgA anti-FLS and RBD levels were significantly less in the group receiving three vaccines than in the hybrid immunity group (Fig. 3A, 3B), they rose to similar levels following breakthrough infection (Fig. 3A, 3B). Omicron breakthrough infection had a substantial impact on the level of plasma IgA against FLS and RBD. There was no significant correlation between IgA anti-FLS levels and ADCC for the hybrid immunity group (Fig. 3C) or for the breakthrough infection group either before or after infection (data not shown), indicating that plasma IgA Abs had no significant impact on the capacity for plasma IgG Abs to elicit ADCC.

Because there was no significant difference between the breakthrough infection and hybrid immunity cohorts in overall IgG levels against SARS-CoV-2 S, we investigated IgG reactivity against S1 and S2 domains and against linear determinants in FLS that we previously showed to be associated with capacity to elicit robust ADCC (30). Recognition of nonneutralizing determinants of S within the S1 and more conserved S2 domains may be critical to this capacity. In the hybrid immunity cohort, Wuhan-Hu-1 infection prior to vaccination skewed SARS-CoV-2 FLS IgG responses toward recognition of the S2 domain compared with dominant anti-S1 IgG responses induced by mRNA-based vaccination (30); therefore, we compared IgG binding to S1 (Fig. 4A) and S2 (Fig. 4B) in plasma from the cohort of individuals with three vaccinations who later experienced Omicron breakthrough infection. After three vaccinations, IgG responses were skewed toward recognition of S1 and remained so despite Omicron breakthrough infection (Fig. 4C). There was no significant change detected in IgG levels against either S1 or S2 at the 1:250 plasma dilution used for ADCC assays following Omicron breakthrough infection (Fig. 4A, 4B). Unlike hybrid immunity, Omicron breakthrough infection does not tilt IgG responses toward recognition of the S2 domain of SARS-CoV-2 FLS (30). A comparison of Ab binding to the three previously identified ADCC-related determinants represented in linear peptides spanning S demonstrated significantly less reactivity attributed to plasma IgG Ab from the cohort with Omicron breakthrough infection than from the hybrid immunity cohort infected prior to vaccination (Fig. 4D–4F). The highest levels of reactivity toward the three determinants associated with ADCC were against those in S1 for the breakthrough infection group, whereas the hybrid immunity group had greater reactivity against the determinant located within S2 (Fig. 4E–4G).

We compared aggregate avidity of IgG anti-S Ab for Wuhan-Hu-1 and Omicron S before and after Omicron breakthrough to assess the impact of Omicron breakthrough infection on anti-S IgG affinity maturation. Prior to breakthrough infection, the avidity of IgG anti-S Ab for the thrice vaccinated individuals was significantly higher against SARS-CoV-2 Wuhan-Hu-1 S than against Omicron S (Fig. 5). Preferred recognition of Wuhan-Hu-1 S was preserved following Omicron breakthrough infection with a selective increase in IgG avidity against Wuhan-Hu-1 S (Fig. 5). The mean aggregate IgG avidity index remained higher against Wuhan-Hu-1 S than against Omicron S after Omicron breakthrough infection, which again illustrates the impact of immune imprinting.

Changes in SARS-CoV-2 S protein receptor binding RBD sequence and structure underlie inadequate neutralization of emerging viruses by Ab raised against S proteins representing earlier variants (3, 11, 14–16, 40). In the absence of strong neutralization, humoral immunity can still contribute protection against severe illness. Nonneutralizing IgG Ab binding conserved determinants in SARS-CoV-2 S can activate antiviral functions through interactions between their Fc region and effector cells bearing appropriate Fc receptors (41). Abs binding to viral Ags expressed on the surface of infected cells engage the CD16 Fc receptor (FcγRIII) on NK cells to trigger cytokine release and killing of virus-infected host cells. Through this mechanism, nonneutralizing Ab can limit viral replication, promote viral clearance, and reduce severity of illness (42–49). Although neutralizing Abs against SARS-CoV-2 target the RBD within S1 and fusion domain within S2 (50, 51), ADCC-eliciting Abs can theoretically target any determinant within S exposed on the infected cell surface. Recent work showed that SARS-CoV-2 S, N, membrane (M) protein, and ORF3a are all targets for ADCC following infection (52), but because the common SARS-CoV-2 vaccine formulations only encode S protein, S is the focus for ADCC studies of hybrid immunity and immunity following breakthrough infection. Capacity for ADCC will depend on the quantity of ADCC-inducing Abs and on a range of qualitative features, including fine specificity and post-translational modification. This study assessed if and how the sequence of SARS-CoV-2 infection versus mRNA vaccination affected IgG and IgA anti-S levels and character, including capacity to elicit ADCC.

A single mRNA vaccination following infection with Wuhan-Hu-1 substantially increased total IgG levels against SARS-CoV-2 S and broadly boosted ADCC capacity across variants (21, 30). In contrast, we found that Omicron breakthrough infection following three mRNA vaccinations had a relatively minor effect on total IgG levels against SARS-CoV-2 S and produced no significant increase over the limited ADCC capacity elicited by three vaccinations. This indicates that the sequence of antigenic exposure through infection versus vaccination and/or the severity of infection markedly influence the developmental pattern and functional quality of humoral immunity. To investigate underlying reasons, we assessed changes in IgG anti-S levels, specificity, and avidity following Omicron breakthrough infection. Breakthrough infection produced a minor increase in IgG anti-S levels apparent at plasma dilution of 1:1000, but not at a lesser dilution of 1:250. Omicron breakthrough infection did significantly increase plasma IgG levels against Wuhan and Omicron RBD and increased plasma IgA levels against Wuhan and Omicron FLS and RBD. Absolute levels of IgG and IgA anti-FLS Abs were not significantly different between the hybrid immunity and breakthrough infection groups after breakthrough infection, despite the distinctly different capacities to elicit ADCC.

The IgG reactivity pattern induced by mRNA vaccination, including preferred recognition of Wuhan-Hu-1 over Omicron S and greater recognition of the S1 over S2 domain, was preserved following Omicron breakthrough infection; however, there was a selective increase in binding to cell surface–expressed Omicron S that had no impact on ADCC. Imprinting of humoral immune responses is generally recognized in the context of variant-specific neutralization, but the impact of imprinting may extend beyond inability to produce high-affinity neutralizing Ab against an infecting variant to affect optimization of Fc-dependent antiviral immunity.

Features associated with robust ADCC elicited by Ab from the hybrid immunity cohort include relative skewing toward recognition of the S2 over S1 domain and recognition of one or more of three linear determinants within S but outside the RBD (30). Because this pattern reflects a focus on more conserved determinants within S, it is also associated with the greater breadth of ADCC than neutralization; however, as with neutralization, ADCC was favored against Wuhan-Hu-1 S over Omicron S-expressing targets (30). Omicron breakthrough infection did little to shift the pattern of humoral immune reactivity toward that associated with broad, robust ADCC. Despite the increase in total IgG binding to plate-bound Wuhan-Hu-1 and Omicron S and a significant increase in IgG binding to cell surface–expressed Omicron S, there was no significant increase in the low-level preexisting ADCC against Omicron or Wuhan-Hu-1 S-expressing target cells. The increase in anti-S IgG was insufficient to affect ADCC, likely because it amplified the extant pattern of reactivity conferring low ADCC capacity. Selectively increased binding to cell surface–expressed Omicron S following breakthrough infection may reflect induction of an IgG population recognizing determinants unique to Omicron S that would be selectively boosted with Omicron S-based vaccines. Although potentially favorable for neutralization of Omicron, this might be detrimental for further development of reactivity against conserved determinants favoring ADCC breadth. A telling feature beyond overall preferred recognition of Wuhan-Hu-1 related to immune imprinting from the original antigenic exposure is the selective increase in IgG avidity for Wuhan-Hu-1 S after Omicron breakthrough infection. We interpret this as further Ag-driven selection and maturation of cross-reactive IgG species induced by Wuhan-Hu-1 with IgG selectively induced by exposure to Omicron S having undergone less maturation such that aggregate IgG avidity against Omicron S was not increased by Omicron breakthrough infection.

Several other studies reported that breakthrough infection increased Ab responses against SARS-CoV-2 S with breakthrough infection after two vaccine doses having roughly the same impact as a third vaccine boost on IgG anti-S levels and neutralization activity (33, 34). In one study, Omicron breakthrough infection after three vaccines resulted in a 46-fold increase in neutralization capacity compared with two vaccines (35). Although we observed increases in IgG and IgA Abs against SARS-CoV-2 RBD after breakthrough infection, the lesser quantitative effects we observed might relate to the number of previous vaccinations with IgG levels reaching a relative plateau after three vaccinations or to the time since vaccination that breakthrough infection occurred. Increased neutralization capacity against Omicron is consistent with further affinity maturation increasing the avidity of cross-neutralizing Ab elicited by exposure to mRNA vaccines encoding Wuhan-Hu-1 S and with a significant increase in plasma IgA Abs following infection. The relationship between plasma and mucosal IgA specificities and levels is not clear, but several studies have reported an association between plasma levels of IgA anti-SARS-CoV-2 S and reduced risk of infection (53–56).

In terms of ADCC capacity, we found that humoral immunity acquired by mRNA vaccination following Wuhan-Hu-1 infection is markedly superior to that acquired by Omicron infection following three mRNA vaccinations. Some question remains whether this reflects initial antigenic exposure through infection versus vaccination or the context of infection, with both preexisting immunity and lesser virulence reducing the severity of Omicron breakthrough infection (57). Although infection with Wuhan-Hu-1 and vaccination with either of the common mRNA vaccines expose the immune system to the same SARS-CoV-2 S protein, the resulting distribution pattern of IgG reactivity across S differs significantly. In the absence of preexisting immunity, SARS-CoV-2 infection is likely to produce higher levels of Ag than mRNA vaccination, and the spread of SARS-CoV-2 through selective cell infection, cell-to-cell transfer, and release into the circulation engenders an overall distinct and differentially localized pattern of Ag processing and presentation to immune cells. Furthermore, because the objective of the immune system is return to equilibrium, once infection occurs, selection for functional specificities that effectively eliminate infection may be increased in comparison with selection for those that prevent infection. This may not substantially happen with Omicron breakthrough infection because of imprinting or because the infection is more rapidly controlled and cleared. A lack of plasma samples from nonvaccinated persons infected with Omicron prevented us from addressing this question directly.

Although breakthrough infection with Omicron does not enhance ADCC capacity and the increase in neutralizing Abs may not have the breadth to address new variants, boosting of vaccine-induced T cell responses against conserved regions of S and induction of new T cell responses against other SARS-CoV-2 proteins have immune benefits. Cellular immune responses are much more conserved across SARS-CoV-2 variants than neutralizing Ab responses and, in concert with Abs that elicit ADCC, should help accelerate viral clearance and reduce the severity of illness when infection occurs (58, 59). Although not yet settled into a seasonal rotation, it is clear that, like influenza, exposure to and repeated infection with SARS-CoV-2 will continue for a large proportion of the population. Other respiratory viruses such as measles with a single serotype and enteroviruses such as hepatitis A with three cross-reactive genotypes induce lifelong immunity following infection. In contrast, the broad diversification of respiratory viruses such as influenza and SARS-CoV-2 enables them to escape neutralizing immunity induced by vaccination and infection, making sterilizing immunity a lofty goal (60–63). In the absence of universal neutralization determinants, even new vaccination strategies inducing mucosal immunity at the site of exposure will lose efficacy due to viral variation.

In conclusion, hybrid immunity from infection prior to vaccination enables a broader and more robust capacity to elicit ADCC than Omicron infection postvaccination. This increased functionality is associated with similar overall IgG anti-FLS levels but differential IgG reactivity across FLS, and it illustrates that immunological imprinting may have impact beyond limiting neutralization of virus variants. Our data suggest that SARS-CoV-2 S-based mRNA vaccination induces an IgG reactivity pattern less favorable for ADCC than the pattern induced by infection and that although boosting total IgG anti-S levels, breakthrough infection sustains this pattern at the expense of boosting ADCC capacity. Given inherent problems with vaccinating to induce neutralizing Ab against viral variants that do not yet exist, vaccine strategies should incorporate induction of robust, more conserved Fc-dependent antiviral functions such as ADCC.

The authors have no financial conflicts of interest.

We thank all participants for providing samples, those who recruited them, and the associated teams. Vector pcDNA3.1(−) containing the SARS-related coronavirus 2, Wuhan-Hu-1 S glycoprotein gene, NR-52420, was contributed by David Veesler, and vector pCMV/R containing the SARS-related coronavirus 2, S glycoprotein gene, lineage B.1.1.529, Omicron variant, NR-56470, was contributed by J. R. Mascola for distribution through Biodefense and Emerging Infections Research Resources Repository, National Institute of Allergy and Infectious Diseases, National Institutes of Health. SARS-related coronavirus 2 S protein peptide array, NR-52402, was obtained through Biodefense and Emerging Infections Research Resources Repository, National Institute of Allergy and Infectious Diseases, National Institutes of Health. The visual abstract was created using BioRender.com, and Adobe Illustrator was used to construct figures.