key: cord-0732876-qfur8tg5
authors: Kaplonek, Paulina; Fischinger, Stephanie; Cizmeci, Deniz; Bartsch, Yannic C.; Kang, Jaewon; Burke, John S.; Shin, Sally A.; Dayal, Diana; Martin, Patrick; Mann, Colin; Amanat, Fatima; Julg, Boris; Nilles, Eric J.; Musk, Elon R.; Menon, Anil S.; Krammer, Florian; Saphire, Erica Ollman; Carfi, Andrea; Alter, Galit
title: mRNA-1273 vaccine-induced antibodies maintain Fc-effector functions across SARS-CoV-2 Variants of Concern
date: 2022-01-06
journal: Immunity
DOI: 10.1016/j.immuni.2022.01.001
sha: 50986d247deeaaae0b809bef910fabc710af5ea0
doc_id: 732876
cord_uid: qfur8tg5

SARS-CoV-2 mRNA vaccines confer robust protection against COVID-19, but the emergence of variants has generated concerns regarding the protective efficacy of currently approved vaccines, which lose neutralizing potency against some variants. Emerging data suggest that antibody functions beyond neutralization may contribute to protection from disease, but little is known about SARS-CoV-2 antibody effector functions. Here we profiled the binding and functional capacity of convalescent antibodies and Moderna mRNA-1273 COVID-19 vaccine-induced antibodies across SARS-CoV-2 variants of concern (VOCs). While neutralizing responses to VOCs decreased in both groups, Fc-mediated responses were distinct. In convalescent individuals, while antibodies exhibited robust binding to VOCs, they showed compromised interactions with Fc-receptors. Conversely, vaccine-induced antibodies also bound robustly to VOCs but continued interacting with Fc-receptors and mediated antibody effector functions. These data point to a resilience in the mRNA vaccine-induced humoral immune response that may continue to protect from SARS-CoV-2 VOCs independent of neutralization.

recently emerging SARS-CoV-2 VOCs on antibody binding and functional humoral immunity induced by natural immunity or the mRNA-1273 vaccine to both the Spike (S) and receptor binding domain (RBD) VOCs. While naturally induced antibodies from convalescent patients bound robustly to the wildtype SARS-CoV-2 Spike and to a slightly lesser degree to VOCs, convalescent VOC-binding antibodies largely failed to interact with Fc-receptors. Conversely, mRNA-1273 induced antibodies bound similarly to wildtype and VOCs, and only demonstrated negligible differences in Fc-receptor engagement, maintaining largely conserved antibody effector functions. These data highlight a previously unappreciated functional gap in immunity in convalescents and point to the resilience of functional mRNA-vaccine-induced antibodies that are likely to continue to bind and confer robust antibody effector functions against VOCs, even in the setting of compromised neutralization.

The emergence and rapid spread of multiple SARS-CoV-2 variants worldwide has raised concerns about the cross-protective activity of mRNA-induced vaccine immunity against these perpetually evolving VOCs. Decreased VOC neutralization has been observed across vaccines (Abdool Karim and de Oliveira, 2021) . However, whether these VOCs also escape other humoral immune antiviral mechanisms remains unclear. To determine how VOCs affect humoral immunity, we profiled convalescent plasma and mRNA vaccine induced humoral immune responses across VOCs. Convalescent plasma samples (n = 305) were collected voluntarily from a community-based seroprevalence study at four sites across the US (California, Texas, North Carolina, and Florida) , where the majority of participants experienced largely asymptomatic/mild symptoms following SARS-CoV-2 infection. Volunteers were followed monthly from May-August 2020, prior to the release of vaccines, and included 19 to 62 years of age, with a prevalence of males (84% males). These profiles were compared to peak immunogenicity timepoints (day 43, two weeks after boosting) from the phase 1 mRNA-1273 clinical trial (ClinicalTrials.gov number, NCT04283461). Eligible vaccine participants (n = 45, with one patient excluded from analysis due to low sample volume (n = 44)) included healthy adults 18 to 55 years of age, with 49% males and 51% females (Jackson et al., 2020) . Limited differences were observed between vaccine doses ( Figure S1) ; therefore, the analysis was performed for all doses combined. Both natural infection and vaccination induce polyclonal pools of antibodies consisting of different isotypes/subclasses and specificities that act synergistically, via the formation of immune complexes, to elicit diverse effector functions (Jefferis et al., 1998) . Several studies have shown differences in antibody titers and neutralization across natural infection and following mRNA vaccination both to the wild type and VOC Spike antigens (Assis et al., 2021 , Jalkanen et al., 2021 , Cho et al., 2021 . However, whether the vaccines further shift the polyclonal humoral immune response remains unclear. Thus, here we aimed to deeply characterize and compare the humoral immune response following SARS-COV-2 infection and COVID-19 mRNA-1273 vaccination across SARS-CoV-2 VOCs to better understand the underlying differences in immune responses and protection against potential infection. Collectively, we observed reduced IgG1, IgG3, IgM, and IgA binding to the B.1.1.7-like (N501YΔ69-70), and E484K (present in B.1.351 and J o u r n a l P r e -p r o o f P1) Spike antigens compared to wildtype D614G Spike in convalescent responses (Figure 1A-B) . Conversely, mRNA-1273 immunized individuals exhibited decreased isotype recognition only for B.1.1.7 VOC compared to the D614G, with robustly conserved or even higher vaccine induced IgG1, IgG3, IgA, and IgM binding across D614G E484K and D614G K417N spike variants ( Figure  1C-D) . No differences in natural and vaccine induced antibody responses were observed between males and females, across age groups, or by symptom severity (Figure S2-S6) . These data point to potential convalescent vulnerabilities in IgG1, IgM, and IgA recognition of the VOCs, but a more limited impact on mRNA vaccine-induced antibody binding to VOCs.

In addition to their ability to bind and block infection, antibodies leverage a wide array of antibody-pathogen functions via antibody constant domain (Fc-domain) interactions with Fcreceptors found on innate immune cells (Lu et al., 2018) . Importantly, Fc-effector function has been linked to protection against several infections, including influenza (Boudreau and Alter, 2019) , malaria , Ebola virus (Meyer et al., 2021) and SARS-CoV-2 . Given our emerging appreciation for a role for Fceffector function in the control/clearance of natural SARS-CoV-2 infection , we explored whether antibodies that evolve in convalescent individuals or mRNA-1273 vaccinees have an equivalent capacity to interact with Fc-receptors. We profiled binding to all low-affinity IgG-Fc-receptors (FcRs) involved in driving IgG effector function ( Figure  2) . Substantial variation was noted in SARS-CoV-2 specific antibody binding to FcRs across VOCs in convalescent plasma (Figure 2A-B) . We observed an overall lower SARS-CoV-2-specific antibody binding to the activating FcR2a in convalescent individuals compared to mRNA vaccinated individuals. Conversely, the opposite pattern was observed for the inhibitory FcR2b receptor, with enhanced binding to FcR2b in convalescent individuals compared to vaccinees. Moreover, compared to the D614G Spike FcR binding, antibodies raised following natural infection exhibited a substantial reduction in binding to FcR2b, FcR3a, and FcR3b to the N501YΔ69-70 Spike variant. Yet, a more prominent loss of FcR binding antibodies was seen for the D614G E484K binding antibodies in convalescent individuals. Importantly, reduced SARS-CoV-2-specific antibody FcR binding was notable, particularly at low convalescent antibody titers. In contrast, convalescent individuals with robust D614G-FcR binding titers exhibited equivalent binding to VOCs. These data point to compromised FcR interactions in convalescent immunity, particularly at low binding titers that may evolve due to mild infection or in the setting of waning immunity (Ibarrondo et al., 2020 , Choe et al., 2021 . Compromised FcR-binding evolved despite largely preserved IgG binding to the VOCs that may render a subset of previously immune individuals, with low antibody titers, vulnerable to re-infection with these new strains.

In contrast to convalescent plasma, mRNA-1273 vaccine induced antibodies exhibited overall stable binding to all activating FcRs, but lower binding to the inhibitory FcR2b ( Figure 2C -D). In comparison to D614G, stronger decline was observed in FcR2a, FcR2b, Fc3a and Fc3b binding to the B.1.1.7 Spike and a trend towards a reduction was noted for the D614G E484K spike for FcR2a and FcR2b binding. However, reduced binding was proportional across binding titer levels, and was not solely observed in individuals with low Spike-specific binding titers. Additionally, no alteration in SARS-CoV-2-specific antibody binding to FcRs was observed for J o u r n a l P r e -p r o o f D614G K417N Spike. These data highlight the disconnect between binding antibodies and FcR engaging antibodies, where Fc-receptor binding was compromised in SARS-CoV-2 Spike-specific antibodies induced following natural infection but not following vaccination. Importantly, it is critical to note that compromised Fc-receptor binding was most clearly observed among convalescent patients with lower antibody titers, whereas convalescent individuals with high titers, approximating levels observed in vaccinees, appeared to bind robustly across the variants. These data point to a functional resilience in mRNA-1273 vaccine-induced antibodies that are less affected by the VOCs compared to low titer convalescent antibodies.

Despite the high efficacy of the mRNA vaccines against the original SARS-CoV-2 variant, waves of variants have emerged that include amino acid substitutions that significantly diminish neutralizing antibody activity (Garcia-Beltran et al., 2021a , Wall et al., 2021b , Planas et al., 2021c attributed to particular mutations in the receptor binding domain (RBD) (Greaney et al., 2021) . However, whether these changes in RBD eliminate all RBD-specific antibody binding and antibody-mediated Fc-receptor interactions has yet to be defined. Here we observed diminished binding of vaccine-induced RBD-specific IgG1 and IgG3 across the B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma) RBDs ( Figure 3A-B) . Less substantial differences were noted for IgM binding across the variants, with a more pronounced loss of IgA binding (Figure 3C-D) . Antibody-FcR binding was compromised across all VOC RBDs, most profoundly for B.1.351 ( Figure 3E-H) . However, FcR interactions with VOC RBDs -were not completely eliminated, but continued to bind in a correlated, albeit dampened, manner compared to antibody binding to the wildtype RBD. These data point to a more profound reduction, but not complete loss, of vaccine induced antibody FcR binding activity to VOC RBDs, despite largely preserved antibody-mediated Fcactivity to the full Spike.

Given the differential FcR binding differences noted across antibodies able to bind to the VOCs, we next sought to determine whether these differences also translated to changes in antibody Fc-effector function across full Spike VOCs for SARS-CoV-2 infected, and mRNA-1273 vaccinated individuals (Figure 4) . Specifically, antibodies from convalescent plasma displayed a prominent reduction in antibody dependent complement deposition (ADCD) for the B.1.351 (beta) variant and similar, but a non-significant, trend for the B.1.1.7 (alpha) variant Spike compared to WT SARS-CoV-2 ( Figure 4A ). Natural antibody mediated complement depositing functions to the P1 VOC Spike antigen were higher than for WT. For mRNA-1273 vaccine-induced SARS-CoV-2 Spikespecific antibodies, only a minor reduction in ADCD was observed for the B.1.1.7 variant, with stable complement-activity to the B.1.351 (beta) variant, and elevated complement fixation for the P1 (gamma) Spike variant compared to WT SARS-CoV-2 ( Figure 4B ). Furthermore, a loss of antibody dependent neutrophil phagocytosis (ADNP) was noted for the B.1.1.7 variant, with a more significant decline in B.1.351 directed ADNP activity in individuals with natural SARS-CoV-2 infection. Similar to ADCD, ADNP activity against the P1 variants was higher than ADNP against the WT SARS-CoV-2 ( Figure 4C) . Conversely, limited to no loss of ADNP activity was observed to the B.1.1.7 and P1 variants in mRNA-1273 vaccinated individuals compared to ADNP activity to the D614G Spike variant. However, a significant, but not complete loss of ADNP was noted to the B.1.351 variant ( Figure 4D ). Antibody dependent cellular phagocytosis (ADCP) was reduced for all VOCs compared to the D614G variant across both convalescent individuals and vaccinated individuals (Figure 4 E-F). Again, these data reinforce the disconnect between the quality and quantity of the humoral immune response to SARS-CoV-2, with equal or enhanced complement and neutrophil phagocytic functions following vaccination to the B.1.1.7 (alpha) and P.1 (gamma) variants but decreased activity to the B.1.351 (beta) variant, but more substantial changes in functional responses to the VOCs following natural infection. Our study reveals some potential nuanced vulnerabilities to emerging VOCs that may help refine our ability to strategically design next-generation vaccines or boosting regimens to provide maximal protection against novel circulating variants of SARS-CoV-2.

Neutralization represents a critical correlate of immunity for many clinically approved vaccines (Plotkin, 2010) . However, protection from infection has been observed: a) just weeks after primary mRNA and Ad26 immunization prior to the induction of neutralizing antibodies , Polack et al., 2020 ; b) in regions of the world where the vaccine neutralizes variants poorly ; as well as c) months after immunization despite declining neutralizing antibody titers (Thompson et al., 2021) , collectively arguing for additional correlates of immunity against SARS-CoV-2. Antibodies may also contribute to protection via various additional mechanisms, through their ability to interact with Fc-receptors or complement (Boudreau and Alter, 2019) . Moreover, Fc-effector functions are emerging as critical correlates of protection in natural SARS-CoV-2 infection as well as in non-human primate vaccine studies ). Yet despite the early high efficacy of approved vaccines, re-infections are common in the setting waning natural and vaccine immunity in the setting of a rise in VOCs (Supasa et al., 2021, Abdool Karim and ). Yet, it is unclear how VOCs disrupt the wholistic natural or vaccine induced humoral immune response, and whether antibody functions, beyond neutralization can compensate for a loss of neutralization.

Unlike neutralizing antibodies that must bind to the Spike at precise sites, antibodies able to elicit Fc-effector function can theoretically target the entire surface of the Spike. Here we demonstrated the disconnect between antibody quantity and quality, measured by FcR binding, to VOCs in convalescent plasma samples, marking unexpected vulnerabilities in the naturally induced humoral immune response to SARS-CoV-2 VOCs. Conversely, mRNA-1273 vaccineinduced Spike-specific antibodies exhibited more robust FcR binding capacity and functionality, pointing to vaccine-induced induction of more resilient antibodies able to target VOCs, bind FcRs, and drive function more flexibly than antibodies that evolve following natural infection. Thus, while neutralizing antibody activity may be selectively compromised to particular variants, the ability of antibodies to continue to leverage the antiviral activity of Fc-effector functions may afford the humoral immune response with the capability of providing persistent opsonophagocytic clearing functions able to attenuate disease should infection occur. Thus, while the ultimate goal of completely blocking SARS-CoV-2 transmission could profoundly accelerate the end of the pandemic, the ability of vaccines to provide protection against disease and death may transform SARS-CoV-2 into a less dangerous virus. The persistence of robust binding, FcR-binding, and Fc-effector function inducing mRNA-vaccine induced Spike-specific antibodies may continue to provide a critical defense against disease caused by emerging VOCs.

Natural resolution of SARS-CoV-2 infection is associated with the generation of a wide variety of different humoral immune titers that persist for several months (Wajnberg et al., 2020) . However, data from Manaus, Brazil, illustrated the limited protective efficacy of naturally generated humoral immune responses against emerging VOCs (Sabino et al., 2021) . Incomplete natural immunity against VOCs may be related to lower quantities of antibodies induced in most convalescent individuals, substantially lower than those induced by mRNA vaccination. Moreover, highly heterogeneous antibody titers were observed among convalescent individuals, that exhibited linked heterogeneous binding capabilities to multiple VOCs, marked by concomitant differences in FcR binding capabilities and Fc-effector functions across VOCs. Importantly, the disconnect between isotype-binding titers and FcR binding was largely observed at lower antibody titers, suggesting that above a particular threshold, even convalescent antibodies may confer a robust barrier against VOCs. Additionally, two doses of mRNA-1273 clearly drove high antibody titers, able to bind to FcRs effectively and drive highly resilient Fceffector function across most VOCs. However, whether additional vaccine platforms will drive equivalent resilient antibody functions and whether additional heterologous prime-boosting strategies may drive robust and durable antibody effector functions remains unclear. Yet, it is likely that autologous or heterologous boosters likely drive robust increases not only in titers but also in FcR binding and function, thereby augmenting both Fab-(neutralization) and Fc-mediated antiviral mechanisms, providing the greatest level of protection against VOCs, even those like Omicron that significantly evade neutralization (Planas et al., 2021b , Garcia-Beltran et al., 2021c , Gruell et al., 2021 .

Thus, despite the loss of neutralization against some VOCs, continued recognition by nonneutralizing mechanisms of protective immunity may be sufficient to confer protection against disease, eliminating the risk of mortality and morbidity previously associated with COVID-19. With emerging deeper phase 3 correlates analyses underway, the precise correlates of immunity will be defined across VOCs, providing critical insights to guide future vaccine design and deployment. The induction of broader, more flexible immune responses that maintain protection against disease and death may ultimately represent a more critical target for global immunity in the face of expected future viral evolution.

There are several limitations to this study. First, we do not know the precise dates of infection for the community-acquired COVID-19 convalescent individuals. However, based on repeat testing, the individuals were no more than three months from infection (Bartsch et al., 2021) following the resolution of acute infection and representing a time point of relative stability in the humoral immune response (Israel et al., 2021 , Wajnberg et al., 2020 . Unfortunately, we were not able to look at long-term vaccine or convalescent durability in this study. However, recent studies have pointed to differential stability in the FcR binding profiles across vaccine platforms, potentially accounting for differences in real-world vaccine efficacy (Puranik et al., 2021 . Second, the convalescence individuals were collected during the wildtype or D614G variant wave, and thus we were unable to profile FcR binding profiles and function elicited by VOC-mediated infections that may vary in their flexibility similar to responses induced by mRNA-1273 vaccination. Third, the mRNA-1273 trial participants were healthy adults, 18 to 55 years of age. Conversely, the convalescent individuals were 19 to 62 years old, thus capturing several individuals a decade older than the vaccinees. Yet, not significant age-effects were observed across each cohort, potentially related to the smaller sample size in these cohorts. Fourth, our community-acquired COVID-19 convalescent individuals experienced largely asymptomatic/mild symptoms following SARS-CoV-2 infection, and no differences were observed across symptom severities. Lastly, antibody-mediated Fc-effector functional assays were performed with cells from healthy donors rather than autologous cells from the convalescent or vaccinated individuals, that may differ and contribute to differential overall capabilities to respond and clear virus upon exposure (Kuri-Cervantes et al., 2020 ). Yet despite these limitations, this study represents the first comprehensive analysis of the additional extraneutralizing antibody properties of mRNA-1273 induced antibodies across VOCs that may provide persisting protection against severe disease and death even with a loss of neutralization.

We thank Nancy Zimmerman, Mark and Lisa Schwartz, an anonymous donor (financial support), Terry and Susan Ragon, and the SAMANA Kay MGH Research Scholars award for their support. We acknowledge support from the Ragon Institute of MGH, MIT, and Harvard, the Massachusetts Consortium on Pathogen Readiness (MassCPR), the NIH (3R37AI080289-11S1, R01AI146785, U19AI42790-01, U19AI135995-02, U19AI42790-01, 1U01CA260476 -01, CIVIC75N93019C00052), the Gates Foundation Global Health Vaccine Accelerator Platform funding (OPP1146996 and INV-001650), Translational Research Institute for Space Health through NASA Cooperative Agreement (NNX16AO69A), and the Musk Foundation. This work used samples from the phase 1 mRNA-1273 study (NCT04283461; DOI: 10.1056/NEJMoa2022483). The mRNA-1273 phase 1 study was sponsored and primarily funded by the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD. This trial has been funded partially with federal funds from the NIAID under grant awards UM1AI148373, to Kaiser Washington; UM1AI148576, UM1AI148684, and NIH P51 OD011132, to Emory University; NIH AID AI149644, and contract award HHSN272201500002C, to Emmes. Funding for the manufacture of mRNA-1273 phase 1 material was provided by the Coalition for Epidemic Preparedness Innovation. The line graph shows the overall FcR-binding profile to the D614G, D614G E484K (black), and the D614G K417N (grey) Spike variants across mRNA-1273 vaccinated individuals. A Pearson correlation was used to establish the strength of the relationship between wildtype and VOC antigen binding. Dots represent the mean value of replicates per serum sample. The fold change was calculated as a ratio of wildtype binding compared to each VOC, indicated in the bracket. Matched nonparametric Friedman test with Dunn's multiple comparisons test was used to calculate the statistical significance for line graphs. Only statistically significant values were shown, and the asterisks represent the adjusted p-values: (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001). correlation was used to establish the strength of the relationship between wildtype and VOC antigen binding. Dots represent the mean value of replicates per serum sample. The fold change was calculated as a ratio of wildtype binding compared to each VOC, indicated in the bracket. Matched nonparametric Friedman test with Dunn's multiple comparisons test was used to calculate the statistical significance for line graphs. Only statistically significant values were shown, and the asterisks represent the adjusted p-values: (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001). Matched nonparametric Friedman test with Dunn's multiple comparisons test was used to calculate the statistical significance for line graphs. Only statistically significant values were shown, and the asterisks represent the adjusted p-values: (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001).

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Galit Alter (galter@mgh.harvard.org).

This study did not generate new unique reagents.

 The dataset generated for the study have been made available in the supplemental material.

 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.

Description of the cohorts mRNA-vaccinated individuals: The phase 1, dose-escalation, open-label clinical trial was designed to determine the safety, reactogenicity, and immunogenicity of mRNA-1273 (mRNA-1273 ClinicalTrials.gov number, NCT04283461). Eligible participants (n = 45) were healthy adults 18 to 55 years of age who received two injections of trial vaccine 28 days apart at a dose of 25 μg (n = 15), 100 μg (n = 15), or 250 μg (n = 15) (Jackson et al., 2020) . One person from a group receiving a dose of 100 μg was excluded from the analysis due to limited sample volume, therefore the analysis was performed with n = 44. The median age of the seropositive population was 32.6 years (range 18-53 years), the gender ratio was 49% male and 51% female, with 86% of non-Hispanic nor Latino and 14% of Hispanic or Latino participants. The study protocol was approved by the Advarra institutional review board. All participants provided written informed consent before enrollment. The trial was conducted under an Investigational New Drug application submitted to the Food and Drug Administration. The vaccine was co-developed by researchers at the National Institute of Allergy and Infectious Diseases (NIAID, the trial sponsor) and at Moderna (Cambridge, MA). The mRNA-1273 vaccine, manufactured by Moderna, encodes the S-2P antigen, consisting of the SARS-CoV-2 glycoprotein with a transmembrane anchor and an intact S1-S2 cleavage site. S-2P was stabilized in its prefusion conformation by two consecutive proline substitutions at amino acid positions 986 and 987, at the top of the central helix in the S2 subunit (Jackson et al., 2020 , Baden et al., 2020 . The mRNA-LNP vaccine (mRNA-1273) was provided as a sterile liquid for injection at a concentration of 0.5 mg per milliliter. Normal saline was used as a diluent to prepare the doses administered. The vaccine was administered as a 0.5 ml injection in the deltoid muscle on days 1 and 29; follow-up visits were scheduled for 7 and 14 days after each vaccination and on days 57, 119, 209, and 394. Only the peak immunogenicity time point (day 43, two weeks following the boost) was included in this study.

Corp.) were volunteer tested for COVID-19, starting in April 2020. Participants completed a study survey including the collection of COVID-19 related symptoms, such as loss of smell/taste, fever, feverish/chills, cough, fatigue, headache, congestion, nausea/vomiting, diarrhea, sore throat, and body/muscle aches. The cohort included mild-symptomatic infections largely (Bartsch et al., 2021) . Specifically, 68% of participant were asymptomatic (0 symptoms reported), 11%, 8%, 6%, 5% and 2% experienced respectively 1, 2, 3, 4 or 5 of mentioned symptoms. Upon obtaining informed consent, blood samples were collected (n = 305) and used for immune profiling. The median age of the seropositive population was 32 years (range 19-62 years), and 84% were males. The enrolled participants were 66% White, 8% Asian, 6% More than one race, 2% Black, 1% American Indian/Alaska Native, and 17% unknown. Volunteers were tested by PCR and for antibodies monthly. All antibody positive individuals were included in the study (Bartsch et al., 2021) .

Fresh peripheral blood was collected by the MGH Blood bank from healthy human volunteers. All volunteers were over 18 years of age, provided written informed consent and all samples were de-identified before use. The studies were approved by the MGH (previously Partners Healthcare) Institutional Review Board. Human NK cells were isolated from fresh peripheral blood and maintained at 37°C, 5% CO2 in RPMI with 10% fetal bovine serum, L-glutamine, penicillin/streptomycin.

THP-1 cells (ATCC) were grown in RPMI-1640 supplemented with 10% fetal bovine serum, Lglutamine, penicillin/streptomycin, HEPES, and beta-mercaptoethanol at 37°C, 5% CO2.

Antibody isotyping and Fcγ-receptor (FcγR) binding were conducted by multiplexed Luminex assay, as previously described (Brown et al., 2012 , Brown et al., 2017 . Antigen-coupled microspheres were washed and incubated with plasma samples at an appropriate sample dilution (1:500 for IgG1 and all low affinity Fcγ-receptors, and 1:100 for all other readouts) for 2 hours at 37°C in 384-well plates (Greiner Bio-One). The high affinity FcR was not tested due to its minimal role in tuning antibody effector function (Nimmerjahn and Ravetch, 2008) . Unbound antibodies were washed away, and antigen-bound antibodies were detected by using a PE-coupled detection antibody for each subclass and isotype (IgG1, IgG3, IgA1, and IgM; Southern Biotech), and Fcγ-receptors were fluorescently labeled with PE before addition to immune complexes (FcγR2a, FcγR3a; Duke Protein Production facility). After one hour of incubation, plates were washed, and flow cytometry was performed with an IQue (Intellicyt), and analysis was performed on IntelliCyt ForeCyt (v8.1). PE median fluorescent intensity (MFI) is reported as a readout for antigen-specific antibody titers.

Antibody-dependent complement deposition (ADCD), antibody-dependent monocyte phagocytosis (ADCP), and antibody-dependent neutrophil phagocytosis (ADNP) were conducted as previously described (Butler et al., 2019 , Karsten et al., 2019 , Fischinger et al., 2019 .

For ADCD, SARS-CoV-2 WT, B.1.1.7, B.1.351, and P1 Spike proteins (LakePharma) were coupled to magnetic Luminex beads (Luminex Corp) by carbodiimide-NHS ester-coupling (Thermo Fisher). Coupled beads were incubated for 2 hours at 37°C with serum samples (1:10 dilution) to form immune complexes and then washed to remove unbound immunoglobulins. In order to measure antibody-dependent deposition of C3, lyophilized guinea pig complement (Cedarlane) was diluted in gelatin veronal buffer with calcium and magnesium (GBV++) (Boston BioProducts) and added to immune complexes. Subsequently, C3 was detected with an anti-C3 fluoresceinconjugated goat IgG fraction detection antibody (Mpbio). The flow cytometry was performed with iQue (Intellicyt) and an S-Lab robot (PAA). ADCD was reported as the median of C3 deposition.

For ADCP and ADNP, SARS-CoV-2 WT, B.1.1.7, B.1.351, and P1 Spike proteins (LakePharma) were biotinylated using EDC (Thermo Fisher) and Sulfo-NHS-LC-LC biotin (Thermo Fisher) and coupled to blue (350/440), crimson (625/645), red-orange (565/580), and yellow-green (505/515) fluorescent Neutravidin-conjugated beads (Thermo Fisher), respectively. To form immune complexes, antigen-coupled beads were incubated for 2 hours at 37°C with 1:100 diluted serum samples and then washed to remove unbound immunoglobulins. For ADCP, the immune complexes were incubated for 16-18 hours with THP-1 cells (25,000 THP-1 cells per well at a concentration of 1.25×10 5 cells/ml in R10 cells/mL) and for ADNP for 1 hour with RBC-lyzed whole blood. Following the incubation, cells were fixed with 4% PFA. For ADNP, For ADNP granulocytes were isolated from whole blood by lysing RBC in ACK lysis buffer (1:10 blood in ACK lysis buffer) for 7 minutes before precipitation by centrifugation. Granulocytes were washed twice with cold PBS and resuspended at 2.5×10 5 cells/ml in R10. 50,000 cells per well were added to each well and incubated with immune complexes for 1 hour at 37°C, 5% CO2. Following the incubation, cells were fixed with 4% PFA. For ADNP, RBC-lyzed whole blood was washed, stained for CD66b+ (Biolegend) to identify neutrophils, and then fixed in 4% PFA. Flow cytometry was performed to identify the percentage of cells that had phagocytosed beads as well as the number of beads that had been phagocytosis (phagocytosis score = % positive cells × Median Fluorescent Intensity of positive cells/10000). The Flow cytometry was performed with 5 Laser LSR Fortessa Flow Cytometer, and analysis was performed using FlowJo V10.7.1.

All Luminex data were log-transformed, and all features were scaled and centered. Statistical analyses were performed using GraphPad Prism 9.0 software. Dots represent the geometric mean fluorescent intensity (gMFI) value of replicates for each serum sample. The relationship of wildtype D614G (x-axis) to the recognition of the VOCs (y-axis) for each Fc-feature was assessed by Pearson correlation. The fold change was calculated as a ratio of wildtype binding compared to each VOC Line graphs show the gMFI per serum sample and the fold change was calculated as a ratio of wildtype binding compared to each VOC. Statistical differences were calculated using matched nonparametric Friedman test with Dunn's multiple comparisons test. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001). J o u r n a l P r e -p r o o f Highlights  mRNA-1273 vaccine induces Spike antibodies able to leverage FcR binding across VOCs  Convalescent Spike antibodies interact but exhibit compromised FcR binding to VOCs  VOCs differentially affect Fc-effector functions in natural infection and vaccination  mRNA-1273-induced antibodies might confer protection independent of neutralization eTOC blurb SARS-CoV-2 mRNA vaccines provide cross-variant protection against COVID-19. Whether this is mediated strictly via neutralization, or whether it is linked to effector functions that may limit, rather block, transmission remains unknown. Kaplonek et al. show that mRNA-1273 vaccinationinduced antibodies preserve Fc-effector responses across variants of concern, whereas antibodies induced following natural infection show compromised interactions with Fcreceptors. .

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

Collaboration between the Fab and Fc contribute to maximal protection against SARS-CoV-2

Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition

2021. mRNA booster immunization elicits potent neutralizing serum activity against the SARS-CoV-2 Omicron variant

Rapid Decay of Anti-SARS-CoV-2 Antibodies in Persons with Mild Covid-19

Large-scale study of antibody titer decay following BNT162b2 mRNA vaccine or SARS-CoV-2 infection

An mRNA Vaccine against SARS-CoV-2

COVID-19 mRNA vaccine induced antibody responses against three SARS-CoV-2 variants

IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation

A versatile high-throughput assay to characterize antibody-mediated neutrophil phagocytosis

Evidence for increased breakthrough rates of SARS-CoV-2 variants of concern

Effectiveness of Covid-19 Vaccines against the B.1.617.2 (Delta) Variant

Beyond binding: antibody effector functions in infectious diseases

Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques

Ebola vaccine-induced protection in nonhuman primates correlates with antibody specificity and Fc-mediated effects

Effectiveness of COVID-19 vaccines against variants of concern in Ontario

Fcgamma receptors as regulators of immune responses

Considerable escape of SARS-CoV-2

Correlates of protection induced by vaccination

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Comparison of two highly-effective mRNA vaccines for COVID-19 during periods of Alpha and Delta variant prevalence

The effect of spike mutations on SARS-CoV-2 neutralization

Resurgence of COVID-19 in Manaus, Brazil, despite high seroprevalence

Mapping functional humoral correlates of protection against malaria challenge following RTS

Robust neutralizing antibodies to SARS-CoV-2 infection persist for months

Neutralising antibody activity against SARS-CoV-2 VOCs B. 1.617. 2 and B. 1.351 by BNT162b2 vaccination

Phenotype and kinetics of SARS-CoV-2-specific T cells in COVID-19 patients with acute respiratory distress syndrome

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

Compromised Humoral Functional Evolution Tracks with SARS-CoV-2 Mortality