Selective SARS-CoV2 BA.2 escape of antibody Fc/Fc-receptor interactions

Summary The number of mutations in the omicron (B.1.1.529) BA.1 variant of concern led to an unprecedented evasion of vaccine induced immunity. However, despite rise in global infections, severe disease did not increase proportionally and is likely linked to persistent recognition of BA.1 by T cells and non-neutralizing opsonophagocytic antibodies. Yet, the emergence of new sublineage BA.2, which is more transmissible than BA.1 despite relatively preserved neutralizing antibody responses, has raised the possibility that BA.2 may evade other vaccine-induced responses. Here, we comprehensively profiled the BNT162b2 vaccine-induced response to several VOCs, including omicron BA.1 and BA.2. While vaccine-induced immune responses were compromised against both omicron sublineages, vaccine-induced antibody isotype titers, and non-neutralizing Fc effector functions were attenuated to the omicron BA.2 spike compared to BA.1. Conversely, FcγR2a and FcγR2b binding was elevated to BA.2, albeit lower than BA.1 responses, potentially contributing to persistent protection against severity of disease.


INTRODUCTION
The rapid and perpetual evolution of SARS-CoV-2 continues to raise concerns for more pathogenic variants able to evade natural or vaccine-induced immune responses. With each wave, SARS-CoV-2 variants of concern (VOCs) have acquired mutations that increased infectivity, either via stabilization of the spike, enhanced binding to the angiotensin converting enzyme-2 (ACE-2), or via alterations in the viral fusion machinery. [1][2][3] Evasion from neutralizing antibodies has progressed in parallel to this evolution due to the natural accumulation of mutations near or around sites involved in infection and fusion. 4,5 The most recent VOCs, omicron, has exhibited the greatest evolutionary leap; acquiring 36 mutations, deletions, or insertions in the spike antigen, omicron is associated with significant evasion of vaccine-induced neutralizing antibodies and increased infectivity. However, despite this remarkable increase in transmissibility, the omicron BA.1 variant has exhibited overall lower pathogenicity and disease. [5][6][7][8] Yet, a new omicron sublineage has emerged, the BA.2 lineage. While BA.2 spike lacks 16 of the alterations characteristic of BA.1, it has acquired 11 additional unique amino acid changes speculated to have increased BA.2 infectivity by 30% over BA.1. 9 The BA.2 lineage has rapidly taken over the epidemic across Southeast Asia, Africa, and across Europe, and the America. 10 Like the parental BA.1, BA.2 evades natural or waning vaccine-induced neutralizing antibodies equally and can be neutralized with boosting. [11][12][13][14][15] While neutralizing, antibodies show minimally changed reactivity from BA.1 to BA.2, we speculated that other immune mechanisms may lose potency against BA.2, thus enabling this lineage to spread more efficiently.
In addition to neutralization, antibodies contribute to infection protection via their ability to use Fc-receptors to leverage the innate immune response and recruit phagocytes. Accumulating data are pointing to a role in Fc effector function in protection against severe COVID-19 infection, monoclonal therapeutic efficacy, and vaccine-mediated protection. [16][17][18][19] Here, we aimed to define whether BA.2 evades these additional functions of the Pfizer BNT162b2 vaccine-induced humoral immune response at peak immunogenicity after the primary series, after eight months, as well as after a boost. We observed significant differences in vaccine-induced immunity to BA. 1

Diminished isotype binding titers to BA.2
Despite nearly identical vaccine-induced neutralizing antibody responses to BA.1 and BA.2, 12 the BA.2 sublineage exhibits a 30% increase in infectiousness and a potential increase in disease severity, 9 calling into question whether this mutant may evade vaccine-induced immunity in a manner that is distinct from the original omicron BA.1 variant. Thus, we probed the ability of Pfizer BNT162b2 vaccine-induced antibodies to bind across VOCs, including the D614G (wild-type), alpha, beta, delta, and omicron BA.1 and BA.2 variants at peak immunogenicity (two weeks after the primary first and second vaccine), after eight months, and two weeks following a BNT162b2 boost (Figures 1, S1, and S2). Binding IgM, IgA, and IgG responses were probed. Robust IgM binding titers were observed across most VOCs after the primary series, albeit responses to BA.1 were lower and lowest to BA.2. IgM responses were rapidly lost over time and were not boosted against the beta, delta, or omicron spikes ( Figure 1A). A similar profile was observed in the IgA response ( Figure 1B), marked by robust IgA levels to the D614G wild-type, alpha, beta, and delta variant, but lower responses to the omicron BA.1 and even lower responses to the BA.2. Moreover, the responses waned over the first eight months proportionally to all variants, but most significantly for the omicron BA. Given the diminished binding profiles to the BA.2 variant, we next aimed to investigate whether this compromised recognition of the BA.2 spike also translated to reduced Fcg-receptor (FcgR) binding. We profiled BNT162b2 vaccine-induced spike-specific FcgR binding across the four low-affinity human FcgRs involved in driving non-neutralizing Fc-effector functions (Figures 2 and S3). Interestingly, binding to the opsonophagocytic activating FcgR2a showed diminished omicron BA.1 and BA.2 reactivity compared to the D614G/wild-type, alpha, beta, and delta variants (Figure 2A). Attenuated omicron BA.1 and BA.2 reactivity persisted with waning and after the boost; however, FcgR2a binding to the BA.2 was not different at post-vaccine timepoints (prime and boost) and remained higher than BA.1 at the waning time point. Conversely, the inhibitory FcgR2b was not different across the omicron sublineages at peak primary 2) variants of concern, and omicron (B.  Figure 2B). Finally, vaccine-induced spike-binding antibodies to the cytotoxicity FcgR3a and neutrophil specific FcgR3b and the IgA binding FcaR showed a similar pattern of lower overall omicron binding responses after the peak primary immune response, although BA.2 exhibited the lowest FcgR3 binding ( Figures 2C-2E). At waning time points both omicron responses elicited substantially lower FcgR3a and FcaR binding than for other VOCs. After boosting, all VOC-specific FcgR3b binding responses increased, although omicron and beta responses increased to a lesser degree (Table 1). Likewise, Fc-receptor binding was highly correlated with antibody titer only at the post-boost time point, highlighting the importance of the third dose in driving coordinated humoral immune responses ( Figure S4). Thus, these data point to conserved activating opsonophagocytic BA.2 specific FcgR2a binding, but marked loss of FcgR2b, FcgR3a, and FcgR3b binding to the BA.2 sublineage.

Vaccine-induced antibodies poorly leverage Fc-effector functions against the BA.2 sublineage spike
Given the observed compromised FcR binding profiles, we aimed to define whether vaccine-induced antibodies could leverage antibody effector functions. At peak immunogenicity after the primary series, BNT162b2-induced spike-specific antibodies drove substantially compromised omicron BA.1 antibodydependent complement deposition (ADCD), neutrophil phagocytosis (ADNP), monocyte phagocytosis (ADCP), and natural killer (NK) cell activation (ADNKA) compared to the D614G spike (Figures 3 and S5). BNT162b2-induced spike-specific functional activity was further compromised to the omicron BA.2 spike. Fc effector activity declined proportionally over time across all of the spike variants, albeit the BA.2 activity remained the lowest. Boosting increased Fc effector activity to all spike variants, although omicron spike responses never reached those of the D614G/Wuhan response, and BA.2 remained the lowest. These data suggest that non-neutralizing antibody effector functions are severely compromised at all timepoints for the BA.2 omicron sublineage, likely resulting in compromised opsonization, FcR activation, and viral clearance to help mitigate disease and transmission.

Multivariate signatures of defective immunity to BA.2
To finally gain a detailed understanding of the fundamental differences in the BNT162b2-induced response to BA.1 and BA.2 that may result in less protection against BA.2, we generated a multivariate partial least squares-discriminant analysis (PLS-DA) model across the two spike antigens at peak immunogenicity after the third dose ( Figure 4). Perfect separation was noted across the BA.1 and BA.2 responses ( Figure 4A), marked by elevated Fc functionality exclusively to the BA.1 lineage. Notably, BA.1-specific immune profiles exhibited a selective enrichment of spike-specific ADCD, ADCP, and ADNP ( Figure 4B). Beyond the overall compromised vaccine-induced immune response to omicron BA.1, vaccine-induced immune responses are further reduced to BA.2, marked by more limited functional IgG/FcgR responses that may lead to poorer control and clearance of this sublineage.

DISCUSSION
Early phase 3 vaccine trial immune correlates analyses, at a time when DG14G/Wuhan and the alpha variant dominated the global pandemic, pointed to the importance of neutralizing antibodies in protection against COVID-19. 20 However, the emergence of the beta, delta, and now omicron VOCs, all of which significantly evade neutralizing antibody responses but do not cause more disease among vaccinated populations, 5-8  21,22 in monoclonal therapeutic activity, [16][17][18][19] and in vaccine mediated protection, 23 here we profiled the Pfizer BNT162b2 functional humoral immune response across VOCs including BA.1 and BA.2. As expected, vaccine induced non-neutralizing antibody responses were lower to omicron variants but were most profoundly diminished against the BA.2 spike antigen. However, selective Fc-receptor binding deficits were noted, marked by maintained/superior FcgR2a/b binding, but reduced FcgR3a/3b binding to BA.2 compared to BA.1. Linked to reduced neutrophil opsonophagocytic activity ( Figure S6), these data point to a selective loss of IgA/neutrophil functional mucosal immunity that may be a key to protection against transmission, but maintenance of FcgR2a/b that may continue to confer protection against disease.
Omicron BA.1 included 36 mutations in the spike antigen that significantly evaded vaccine-induced and monoclonal therapeutic recognition. 5 Similarly, to the loss of neutralizing antibody responses, Moderna mRNA1273, Pfizer BNT162b2, and CoronaVac vaccine induced immune responses lost recognition of receptor binding domain (RBD) but maintained robust recognition and non-neutralizing antibody responses to the BA.1 spike antigen. 24 Conversely, BA.2 includes 11 additional spike mutations that appear to further compromise antibody binding to the spike, resulting in compromised Fc-receptor binding and significantly lower non-neutralizing antibody responses to this new sublineage. Whether these additional 11 mutations result in disruption of immune complex density, or create geometric changes in the spike, precluding the formation of robust immune complexes and binding to particular Fc-receptors remains incompletely understood but could provide key insights for the design of next generation vaccine-antigen inserts to maximize the induction of highly functional antibodies that, in addition to neutralization, may be a key to protection against transmission. Thus, it will be critical to map the precise functional epitope footprints of protective antibodies to guide vaccine design in the future.
Compared to our previous findings of deficits in omicron-specific VOCs-specific Fc effector activity in mRNA vaccine recipients, the data presented here point to a more significant loss of BA.2 specific Fc effector function. 25 Furthermore, direct comparison of BNT162b2 immunity to BA.1 and BA.2 pointed to selective defects in the BA.2-specific response, marked by a global loss of FcgR dependent effector responses, and linked to a specific loss of FcgR2b, FcgR3a, and FcgR3b binding. Moreover, we observed a titer-independent loss of complement fixing activity, potentially related to the loss of IgM binding to omicron, as well as to geometric differences in IgG recognition of the mutated omicron sublineage spikes that may potentially preclude complement binding, the key for cytotoxic destruction, induction of opsonophagocytosis, and T cell immunity. 26,27 Moreover, coupled to the quantitative loss of opsonophagocytic and cytotoxic functions, these data point to a unique axis of immunity that may be lost to BA.2. Specifically, neutrophils and monocytes respond to both IgG1/3 immune complexes via antibody binding to FcgR2a and FcgR3b, IgM and IgA-formed immune complexes via the constitutive expression of FcmR 28 or FcaR, 29,30 and via complement receptors, 31 enabling phagocytes to rapidly clear immune complexes both systemically, as well as at mucosal membranes. In contrast to this compromised Fc effector activity, neutralizing iScience Article antibody responses were similar across omicron sublineages. 12,13 Additionally, while neutralization is also highly dependent on antibody titers, this activity is traditionally reported in a titer-uncorrected manner. However, upon titer correction of Fc effector data, we observed a strong relationship between antibody binding differences (titer) and ADCP, ADNP, and ADNKA, but enhanced complement fixing defects even after titer correction. These data suggest that reduced BA.2 cross-reactivity induced binding defects may contribute to reduced Fc effector mediated control of this omicron sublineage, further compounded by compromised complement activity. On a per-antibody level, no difference in BA.1 or BA.2 specific antibodies that elicit ADNP, ADCP or ADNKA were observed. Although, BA.1 specific antibodies were more prone to induce Fc-receptor independent complement deposition (ADCD) than BA.2 antibodies on the post second and third dose time points ( Figure S7). Thus, the global loss Fc effector activity against BA.2 may point to a selective deficit in humoral BA.2 specific immunity related to both quantitative and qualitative changes in antibody recognition of the VOCs. 2) variants of concern, and omicron (B.   , nuanced binding has been observed across the newer sublineages that likely account for further evasion of Fc effector function. Moreover, this study did not explore functional humoral immunity to BA.2 following additional vaccine platforms, yet increased omicron sublineage transmission appears to occur globally in a vaccine platform independent manner, even after boosting, 38 suggesting that promoting more wild-type-specific spike immunity may be insufficient to drive robust antibody effector functions against these rapidly evolving variants. However, while boosting with omicron spike led to a marginal increase in neutralization across VOCs compared to the wild-type antigen, these studies did not take into consideration the critical nature of VOCs breadth of binding for shaping Fc effector function. Thus, future efforts focusing on the importance of VOCs based vaccination may unravel the critical nature of vaccine-induced breadth on shaping additional antibody effector responses that may be a key to protection against severity of disease, rather than simple blockade of transmission. Linked to future quantitative assessments of Fc effector levels, rather than qualitative measurements presented here, thresholds of protective immunity may be defined to guide future boosting recommendations.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Materials availability
This study did not generate new unique reagents.

Data and code availability
All relevant data is reported in this paper.
This paper does not report original code.
Any additional information or raw data will be shared by the lead contact upon reasonable request.

Study population
Plasma samples from a total of 24 BNT162b2 vaccinated individuals (median age: 34 years, range: 23-69 years, 88 % female) were obtained from a specimen biorepository at Beth Israel Deaconess Medical Center (BIDMC). Participants received three doses of 30 mg BNT162b2. The first doses were given approx. 21 days apart as per manufacturers recommendation. The third dose was given a median 254 days (range 248-258 days) after the second dose ( Figure S1). Samples for all three timepoints were available for 13 individuals while for three included individuals only the post 2 nd and post 3 rd dose timepoint and for one individual the pre-and post 3 rd timepoint was available. Additionally, two individuals with only the post 2 nd dose and four individuals with the post 3 rd dose timepoint were included in the analysis. The individuals that did not have a first or second sample were all age and gender matched and did not have any reported co-morbidities. All participants provided informed consent prior to enrollment into the study. No participant reported or had serological evidence (Nucleocapsid-specific antibody titer) of previous SARS-CoV-2 infection, received other COVID-19 vaccines, or immunosuppressive medications. This study was overseen and approved by the BIDMC Institutional Review Board (#2020P000361) and the MassGeneral Institutional Review Board (#2021P002628).

Antigens and biotinylation
Spike protein antigens for the D614G wildtype, alpha (B.1.1.7), beta (B.1.351), and delta (B.1.617.2) VOCs were obtained from Sino-Biologicals. Omicron (B.1.1.529) BA1 and BA2 Spikes were produced in house. 39 All antigens were produced in mammalian HEK293 cells. A strep-tag for purification was added to the C-terminus of the Omicron Spikes, whereas all other Spike variants had a His-tag at the C-terminus. All Spike antigens were expressed in the HexaPro (S-2P) form to stabilize the prefusion state of the protein.
For functional assays all antigens were biotinylated using an NHS-Sulfo-LC-LC kit according to the manufacturer's instruction (Thermo Fisher Scientific). Excessive biotin was removed by size exclusion chromatography using Zeba-Spin desalting columns (7kDa cutoff, Thermo Fisher Scientific).