Mucosal antibody responses following Vaxzevria vaccination.

Mucosal antibodies play a key role in protection against breakthrough COVID-19 infections and emerging viral variants. Intramuscular adenovirus-based vaccination (Vaxzevria) only weakly induces nasal IgG and IgA responses, unless vaccinees have been previously infected. However, little is known about how Vaxzevria vaccination impacts the ability of mucosal antibodies to induce Fc responses, particularly against SARS-CoV-2 variants of concern (VoCs). Here, we profiled paired mucosal (saliva, tears) and plasma antibodies from COVID-19 vaccinated only vaccinees (uninfected, vaccinated) and COVID-19 recovered vaccinees (COVID-19 recovered, vaccinated) who both received Vaxzevria vaccines. SARS-CoV-2 ancestral-specific IgG antibodies capable of engaging FcγR3a were significantly higher in the mucosal samples of COVID-19 recovered Vaxzevria vaccinees in comparison with vaccinated only vaccinees. However, when IgG and FcγR3a engaging antibodies were tested against a panel of SARS-CoV-2 VoCs, the responses were ancestral-centric with weaker recognition of Omicron strains observed. In contrast, salivary IgA, but not plasma IgA, from Vaxzevria vaccinees displayed broad cross-reactivity across all SARS-CoV-2 VoCs tested. Our data highlight that while intramuscular Vaxzevria vaccination can enhance mucosal antibodies responses in COVID-19 recovered vaccinees, restrictions by ancestral-centric bias may have implications for COVID-19 protection. However, highly cross-reactive mucosal IgA could be key in addressing these gaps in mucosal immunity and may be an important focus of future SARS-CoV-2 vaccine development.

Antibody Fc-binding profiles and ACE2 affinity to SARS-CoV-2 RBD variants.

Emerging SARS-CoV-2 variants, notably Omicron, continue to remain a formidable challenge to worldwide public health. The SARS-CoV-2 receptor-binding domain (RBD) is a hotspot for mutations, reflecting its critical role at the ACE2 interface during viral entry. Here, we comprehensively investigated the impact of RBD mutations, including 5 variants of concern (VOC) or interest-including Omicron (BA.2)-and 33 common point mutations, both on IgG recognition and ACE2-binding inhibition, as well as FcγRIIa- and FcγRIIIa-binding antibodies, in plasma from two-dose BNT162b2-vaccine recipients and mild-COVID-19 convalescent subjects obtained during the first wave using a custom-designed bead-based 39-plex array. IgG-recognition and FcγR-binding antibodies were decreased against the RBD of Beta and Omicron, as well as point mutation G446S, found in several Omicron sub-variants as compared to wild type. Notably, while there was a profound decrease in ACE2 inhibition against Omicron, FcγR-binding antibodies were less affected, suggesting that Fc functional antibody responses may be better retained against the RBD of Omicron in comparison to neutralization. Furthermore, while measurement of RBD-ACE2-binding affinity via biolayer interferometry showed that all VOC RBDs have enhanced affinity to human ACE2, we demonstrate that human ACE2 polymorphisms, E35K (rs1348114695) has reduced affinity to VOCs, while K26R (rs4646116) and S19P (rs73635825) have increased binding kinetics to the RBD of VOCs, potentially affecting virus-host interaction and, thereby, host susceptibility. Collectively, our findings provide in-depth coverage of the impact of RBD mutations on key facets of host-virus interactions.

Serum IgA Fc effector functions in infectious disease and cancer.

Immunoglobulin (Ig) A is the most abundant antibody isotype present at mucosal surfaces and the second most abundant in human serum. In addition to preventing pathogen entry at mucosal surfaces, IgA can control and eradicate bacterial and viral infections through a variety of antibody-mediated innate effector cell mechanisms. The role of mucosal IgA in infection (e.g. neutralization) and in inflammatory homeostasis (e.g. allergy and autoimmunity) has been extensively investigated; by contrast, serum IgA is comparatively understudied. IgA binding to fragment crystallizable alpha receptor plays a dual role in the activation and inhibition of innate effector cell functions. Mounting evidence suggests that serum IgA induces potent effector functions against various bacterial and some viral infections including Neisseria meningitidis and rotavirus. Furthermore, in the era of immunotherapy, serum IgA provides an interesting alternative to classical IgG monoclonal antibodies to treat cancer and infectious pathogens. Here we discuss the role of serum IgA in infectious diseases with reference to bacterial and viral infections and the potential for IgA as a monoclonal antibody therapy.

Preexisting immunity restricts mucosal antibody recognition of SARS-CoV-2 and Fc profiles during breakthrough infections.

Understanding mucosal antibody responses from SARS-CoV-2 infection and/or vaccination is crucial to develop strategies for longer term immunity, especially against emerging viral variants. We profiled serial paired mucosal and plasma antibodies from COVID-19 vaccinated only vaccinees (vaccinated, uninfected), COVID-19-recovered vaccinees (recovered, vaccinated), and individuals with breakthrough Delta or Omicron BA.2 infections (vaccinated, infected). Saliva from COVID-19-recovered vaccinees displayed improved antibody-neutralizing activity, Fcγ receptor (FcγR) engagement, and IgA levels compared with COVID-19-uninfected vaccinees. Furthermore, repeated mRNA vaccination boosted SARS-CoV-2-specific IgG2 and IgG4 responses in both mucosa biofluids (saliva and tears) and plasma; however, these rises only negatively correlated with FcγR engagement in plasma. IgG and FcγR engagement, but not IgA, responses to breakthrough COVID-19 variants were dampened and narrowed by increased preexisting vaccine-induced immunity against the ancestral strain. Salivary antibodies delayed initiation following breakthrough COVID-19 infection, especially Omicron BA.2, but rose rapidly thereafter. Importantly, salivary antibody FcγR engagements were enhanced following breakthrough infections. Our data highlight how preexisting immunity shapes mucosal SARS-CoV-2-specific antibody responses and has implications for long-term protection from COVID-19.

Heterologous SARS-CoV-2 IgA neutralising antibody responses in convalescent plasma.

Objectives: Following infection with SARS-CoV-2, virus-specific antibodies are generated, which can both neutralise virions and clear infection via Fc effector functions. The importance of IgG antibodies for protection and control of SARS-CoV-2 has been extensively reported. By comparison, other antibody isotypes including IgA have been poorly characterised. Methods: Here, we characterised plasma IgA from 41 early convalescent COVID-19 subjects for neutralisation and Fc effector functions. Results: Convalescent plasma IgA from > 60% of the cohort had the capacity to inhibit the interaction between wild-type RBD and ACE2. Furthermore, a third of the cohort induced stronger IgA-mediated ACE2 inhibition than matched IgG when tested at equivalent concentrations. Plasma IgA and IgG from this cohort broadly recognised similar RBD epitopes and had similar capacities to inhibit ACE2 from binding to 22 of the 23 prevalent RBD mutations assessed. However, plasma IgA was largely incapable of mediating antibody-dependent phagocytosis in comparison with plasma IgG. Conclusion: Overall, convalescent plasma IgA contributed to the neutralising antibody response of wild-type SARS-CoV-2 RBD and various RBD mutations. However, this response displayed large heterogeneity and was less potent than IgG.

Tear antibodies to SARS-CoV-2: implications for transmission.

OBJECTIVES: SARS-CoV-2 can be transmitted by aerosols, and the ocular surface may be an important route of transmission. Little is known about protective antibody responses to SARS-CoV-2 in tears after infection or vaccination. We analysed the SARS-CoV-2-specific IgG and IgA responses in human tears after either COVID-19 infection or vaccination. METHODS: We measured the antibody responses in 16 subjects with COVID-19 infection for an average of 7 months before, and 15 subjects before and 2 weeks post-Comirnaty (Pfizer-BioNtech) vaccination. Plasma, saliva and basal tears were collected. Eleven pre-pandemic individuals were included as healthy controls. RESULTS: IgG antibodies to spike and nucleoprotein were detected in tears, saliva and plasma from subjects with prior SARS-CoV-2 infection in comparison with uninfected controls. While receptor-binding domain (RBD)-specific antibodies were detected in plasma, minimal RBD-specific antibodies were detected in tears and saliva. By contrast, high levels of IgG antibodies to spike and RBD, but not nucleoprotein, were induced in tears, saliva and plasma of subjects receiving 2 doses of the Comirnaty vaccine. Increased levels of IgA1 and IgA2 antibodies to SARS-CoV-2 antigens were detected in plasma following infection or vaccination but were unchanged in tears and saliva. Comirnaty vaccination induced high neutralising Abs in the plasma, but limited neutralising antibodies were detected in saliva or tears. CONCLUSION: Both infection and vaccination induce SARS-CoV-2-specific IgG antibodies in tears. RBD-specific IgG antibodies in tears were induced by vaccination but were not present 7 months post-infection. This suggests the neutralising antibodies may be low in the tears late following infection.

Decay of Fc-dependent antibody functions after mild to moderate COVID-19.

The capacity of antibodies to engage with immune cells via the Fc region is important in preventing and controlling many infectious diseases. The evolution of such antibodies during convalescence from coronavirus disease 2019 (COVID-19) is largely unknown. We develop assays to measure Fc-dependent antibody functions against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-expressing cells in serial samples from subjects primarily with mild-moderate COVID-19 up to 149 days post-infection. We find that S-specific antibodies capable of engaging Fcγ receptors decay over time, with S-specific antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADP) activity within plasma declining accordingly. Although there is significant decay in ADCC and ADP activity, they remain readily detectable in almost all subjects at the last time point studied (94%) in contrast with neutralization activity (70%). Although it remains unclear the degree to which Fc effector functions contribute to protection against SARS-CoV-2 re-infection, our results indicate that antibodies with Fc effector functions persist longer than neutralizing antibodies.

Simultaneous evaluation of antibodies that inhibit SARS-CoV-2 variants via multiplex assay.

The SARS-CoV-2 receptor binding domain (RBD) is both the principal target of neutralizing antibodies and one of the most rapidly evolving domains, which can result in the emergence of immune escape mutations, limiting the effectiveness of vaccines and antibody therapeutics. To facilitate surveillance, we developed a rapid, high-throughput, multiplex assay able to assess the inhibitory response of antibodies to 24 RBD natural variants simultaneously. We demonstrate how this assay can be implemented as a rapid surrogate assay for functional cell-based serological methods to measure the SARS-CoV-2 neutralizing capacity of antibodies at the angiotensin-converting enzyme 2-RBD (ACE2-RBD) interface. We describe the enhanced affinity of RBD variants N439K, S477N, Q493L, S494P, and N501Y to the ACE2 receptor and demonstrate the ability of this assay to bridge a major gap for SARS-CoV-2 research, informing selection of complementary monoclonal antibody candidates and the rapid identification of immune escape to emerging RBD variants following vaccination or natural infection.

Evolution of immune responses to SARS-CoV-2 in mild-moderate COVID-19.

The durability of infection-induced SARS-CoV-2 immunity has major implications for reinfection and vaccine development. Here, we show a comprehensive profile of antibody, B cell and T cell dynamics over time in a cohort of patients who have recovered from mild-moderate COVID-19. Binding and neutralising antibody responses, together with individual serum clonotypes, decay over the first 4 months post-infection. A similar decline in Spike-specific CD4+ and circulating T follicular helper frequencies occurs. By contrast, S-specific IgG+ memory B cells consistently accumulate over time, eventually comprising a substantial fraction of circulating the memory B cell pool. Modelling of the concomitant immune kinetics predicts maintenance of serological neutralising activity above a titre of 1:40 in 50% of convalescent participants to 74 days, although there is probably additive protection from B cell and T cell immunity. This study indicates that SARS-CoV-2 immunity after infection might be transiently protective at a population level. Therefore, SARS-CoV-2 vaccines might require greater immunogenicity and durability than natural infection to drive long-term protection.

Systems serology detects functionally distinct coronavirus antibody features in children and elderly.

The hallmarks of COVID-19 are higher pathogenicity and mortality in the elderly compared to children. Examining baseline SARS-CoV-2 cross-reactive immunological responses, induced by circulating human coronaviruses (hCoVs), is needed to understand such divergent clinical outcomes. Here we show analysis of coronavirus antibody responses of pre-pandemic healthy children (n = 89), adults (n = 98), elderly (n = 57), and COVID-19 patients (n = 50) by systems serology. Moderate levels of cross-reactive, but non-neutralizing, SARS-CoV-2 antibodies are detected in pre-pandemic healthy individuals. SARS-CoV-2 antigen-specific Fcγ receptor binding accurately distinguishes COVID-19 patients from healthy individuals, suggesting that SARS-CoV-2 infection induces qualitative changes to antibody Fc, enhancing Fcγ receptor engagement. Higher cross-reactive SARS-CoV-2 IgA and IgG are observed in healthy elderly, while healthy children display elevated SARS-CoV-2 IgM, suggesting that children have fewer hCoV exposures, resulting in less-experienced but more polyreactive humoral immunity. Age-dependent analysis of COVID-19 patients, confirms elevated class-switched antibodies in elderly, while children have stronger Fc responses which we demonstrate are functionally different. These insights will inform COVID-19 vaccination strategies, improved serological diagnostics and therapeutics.