The BNT162b2 mRNA SARS-CoV-2 vaccine induces transient afucosylated IgG1 in naive but not in antigen-experienced vaccinees.

BACKGROUND: Afucosylated IgG1 responses have only been found against membrane-embedded epitopes, including anti-S in SARS-CoV-2 infections. These responses, intrinsically protective through enhanced FcγRIIIa binding, can also trigger exacerbated pro-inflammatory responses in severe COVID-19. We investigated if the BNT162b2 SARS-CoV-2 mRNA also induced afucosylated IgG responses. METHODS: Blood from vaccinees during the first vaccination wave was collected. Liquid chromatography-Mass spectrometry (LC-MS) was used to study anti-S IgG1 Fc glycoprofiles. Responsiveness of alveolar-like macrophages to produce proinflammatory cytokines in presence of sera and antigen was tested. Antigen-specific B cells were characterized and glycosyltransferase levels were investigated by Fluorescence-Activated Cell Sorting (FACS). FINDINGS: Initial transient afucosylated anti-S IgG1 responses were found in naive vaccinees, but not in antigen-experienced ones. All vaccinees had increased galactosylated and sialylated anti-S IgG1. Both naive and antigen-experienced vaccinees showed relatively low macrophage activation potential, as expected, due to the low antibody levels for naive individuals with afucosylated IgG1, and low afucosylation levels for antigen-experienced individuals with high levels of anti-S. Afucosylation levels correlated with FUT8 expression in antigen-specific plasma cells in naive individuals. Interestingly, low fucosylation of anti-S IgG1 upon seroconversion correlated with high anti-S IgG levels after the second dose. INTERPRETATION: Here, we show that BNT162b2 mRNA vaccination induces transient afucosylated anti-S IgG1 responses in naive individuals. This observation warrants further studies to elucidate the clinical context in which potent afucosylated responses would be preferred. FUNDING: LSBR1721, 1908; ZonMW10430012010021, 09150161910033, 10430012010008; DFG398859914, 400912066, 390884018; PMI; DOI4-Nr. 3; H2020-MSCA-ITN 721815.

Immunoassay for quantification of antigen-specific IgG fucosylation.

BACKGROUND: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (FcγR). Absence of fucose on the highly conserved N-linked glycan in the IgG Fc domain strongly enhances IgG binding and activation of myeloid and natural killer (NK) cell FcγRs. Although afucosylated IgG can provide increased protection (malaria and HIV), it also boosts immunopathologies in alloimmune diseases, COVID-19 and dengue fever. Quantifying IgG fucosylation currently requires sophisticated methods such as liquid chromatography-mass spectrometry (LC-MS) and extensive analytical skills reserved to highly specialized laboratories. METHODS: Here, we introduce the Fucose-sensitive Enzyme-linked immunosorbent assay (ELISA) for Antigen-Specific IgG (FEASI), an immunoassay capable of simultaneously quantitating and qualitatively determining IgG responses. FEASI is a two-tier immunoassay; the first assay is used to quantify antigen-specific IgG (IgG ELISA), while the second gives FcγRIIIa binding-dependent readout which is highly sensitive to both the IgG quantity and the IgG Fc fucosylation (FcγR-IgG ELISA). FINDINGS: IgG Fc fucosylation levels, independently determined by LC-MS and FEASI, in COVID-19 responses to the spike (S) antigen, correlated very strongly by simple linear regression (R2=0.93, p < 0.0001). The FEASI method was then used to quantify IgG levels and fucosylation in COVID-19 convalescent plasma which was independently validated by LC-MS. INTERPRETATION: FEASI can be reliably implemented to measure relative and absolute IgG Fc fucosylation and quantify binding of antigen-specific IgG to FcγR in a high-throughput manner accessible to all diagnostic and research laboratories. FUNDING: This work was funded by the Stichting Sanquin Bloedvoorziening (PPOC 19-08 and SQI00041) and ZonMW 10430 01 201 0021.

Immunoglobulin G1 Fc glycosylation as an early hallmark of severe COVID-19.

BACKGROUND: Immunoglobulin G1 (IgG1) effector functions are impacted by the structure of fragment crystallizable (Fc) tail-linked N-glycans. Low fucosylation levels on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein-specific IgG1 has been described as a hallmark of severe coronavirus disease 2019 (COVID-19) and may lead to activation of macrophages via immune complexes thereby promoting inflammatory responses, altogether suggesting involvement of IgG1 Fc glycosylation modulated immune mechanisms in COVID-19. METHODS: In this prospective, observational single center cohort study, IgG1 Fc glycosylation was analyzed by liquid chromatography-mass spectrometry following affinity capturing from serial plasma samples of 159 SARS-CoV-2 infected hospitalized patients. FINDINGS: At baseline close to disease onset, anti-S IgG1 glycosylation was highly skewed when compared to total plasma IgG1. A rapid, general reduction in glycosylation skewing was observed during the disease course. Low anti-S IgG1 galactosylation and sialylation as well as high bisection were early hallmarks of disease severity, whilst high galactosylation and sialylation and low bisection were found in patients with low disease severity. In line with these observations, anti-S IgG1 glycosylation correlated with various inflammatory markers. INTERPRETATION: Association of low galactosylation, sialylation as well as high bisection with disease severity and inflammatory markers suggests that further studies are needed to understand how anti-S IgG1 glycosylation may contribute to disease mechanism and to evaluate its biomarker potential. FUNDING: This project received funding from the European Commission's Horizon2020 research and innovation program for H2020-MSCA-ITN IMforFUTURE, under grant agreement number 721815, and supported by Crowdfunding Wake Up To Corona, organized by the Leiden University Fund.

Saliva SARS-CoV-2 Antibody Prevalence in Children.

COVID-19 patients produce circulating and mucosal antibodies. In adults, specific saliva antibodies have been detected. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We therefore assessed SARS-CoV-2-specific antibody prevalence in serum and saliva in children in the Netherlands. We assessed SARS-CoV-2 antibody prevalence in serum and saliva of 517 children attending medical services in the Netherlands (irrespective of COVID-19 exposure) from April to October 2020. The prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N)-specific IgG and IgA were evaluated with an exploratory Luminex assay in serum and saliva and with the Wantai SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay in serum. Using the Wantai assay, the RBD-specific antibody prevalence in serum was 3.3% (95% confidence interval [CI]. 1.9 to 5.3%). With the Luminex assay, we detected heterogeneity between antibodies for S, RBD, and N antigens, as IgG and IgA prevalence ranged between 3.6 and 4.6% in serum and between 0 and 4.4% in saliva. The Luminex assay also revealed differences between serum and saliva, with SARS-CoV-2-specific IgG present in saliva but not in serum for 1.5 to 2.7% of all children. Using multiple antigen assays, the IgG prevalence for at least two out of three antigens (S, RBD, or N) in serum or saliva can be calculated as 3.8% (95% CI, 2.3 to 5.6%). Our study displays the heterogeneity of the SARS-CoV-2 antibody response in children and emphasizes the additional value of saliva antibody detection and the combined use of different antigens. IMPORTANCE Comprehending humoral immunity to SARS-CoV-2, including in children, is crucial for future public health and vaccine strategies. Others have suggested that mucosal antibody measurement could be an important and more convenient tool to evaluate humoral immunity compared to circulating antibodies. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We show the heterogeneity of SARS-CoV-2 antibodies, in terms of both antigen specificity and differences between circulating and mucosal antibodies, emphasizing the additional value of saliva antibody detection next to detection of antibodies in serum.

Dynamics of antibodies to SARS-CoV-2 in convalescent plasma donors.

OBJECTIVES: Characterisation of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT-PCR-positive SARS-CoV-2 convalescent adults during the first 250 days after onset of symptoms. METHODS: We measured antibody responses to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the nucleocapsid protein in 844 longitudinal samples from 151 RT-PCR-positive SARS-CoV-2 convalescent adults. With a median of 5 (range 2-18) samples per individual, this allowed quantitative analysis of individual longitudinal antibody profiles. Kinetic profiles were analysed by mixed-effects modelling. RESULTS: All donors were seropositive at the first sampling moment, and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels declined with median half-lives of 62 and 59 days, respectively, 2-5 months after symptom onset, and several-fold variation in half-lives of individuals was observed. The rate of decline of antibody levels diminished during extended follow-up, which points towards long-term immunological memory. The magnitude of the anti-RBD IgG response correlated well with neutralisation capacity measured in a classic plaque reduction assay and in an in-house developed competitive assay. CONCLUSION: The result of this study gives valuable insight into the long-term longitudinal response of antibodies to SARS-CoV-2.

High titers and low fucosylation of early human anti-SARS-CoV-2 IgG promote inflammation by alveolar macrophages.

Patients diagnosed with coronavirus disease 2019 (COVID-19) become critically ill primarily around the time of activation of the adaptive immune response. Here, we provide evidence that antibodies play a role in the worsening of disease at the time of seroconversion. We show that early-phase severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) spike protein-specific immunoglobulin G (IgG) in serum of critically ill COVID-19 patients induces excessive inflammatory responses by human alveolar macrophages. We identified that this excessive inflammatory response is dependent on two antibody features that are specific for patients with severe COVID-19. First, inflammation is driven by high titers of anti-spike IgG, a hallmark of severe disease. Second, we found that anti-spike IgG from patients with severe COVID-19 is intrinsically more proinflammatory because of different glycosylation, particularly low fucosylation, of the antibody Fc tail. Low fucosylation of anti-spike IgG was normalized in a few weeks after initial infection with SARS-CoV-2, indicating that the increased antibody-dependent inflammation mainly occurs at the time of seroconversion. We identified Fcγ receptor (FcγR) IIa and FcγRIII as the two primary IgG receptors that are responsible for the induction of key COVID-19-associated cytokines such as interleukin-6 and tumor necrosis factor. In addition, we show that anti-spike IgG-activated human macrophages can subsequently break pulmonary endothelial barrier integrity and induce microvascular thrombosis in vitro. Last, we demonstrate that the inflammatory response induced by anti-spike IgG can be specifically counteracted by fostamatinib, an FDA- and EMA-approved therapeutic small-molecule inhibitor of Syk kinase.

Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity.

Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.

Development of a SARS-CoV-2 Total Antibody Assay and the Dynamics of Antibody Response over Time in Hospitalized and Nonhospitalized Patients with COVID-19.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infections often cause only mild disease that may evoke relatively low Ab titers compared with patients admitted to hospitals. Generally, total Ab bridging assays combine good sensitivity with high specificity. Therefore, we developed sensitive total Ab bridging assays for detection of SARS-CoV-2 Abs to the receptor-binding domain (RBD) and nucleocapsid protein in addition to conventional isotype-specific assays. Ab kinetics was assessed in PCR-confirmed, hospitalized coronavirus disease 2019 (COVID-19) patients (n = 41) and three populations of patients with COVID-19 symptoms not requiring hospital admission: PCR-confirmed convalescent plasmapheresis donors (n = 182), PCR-confirmed hospital care workers (n = 47), and a group of longitudinally sampled symptomatic individuals highly suspect of COVID-19 (n = 14). In nonhospitalized patients, the Ab response to RBD is weaker but follows similar kinetics, as has been observed in hospitalized patients. Across populations, the RBD bridging assay identified most patients correctly as seropositive. In 11/14 of the COVID-19-suspect cases, seroconversion in the RBD bridging assay could be demonstrated before day 12; nucleocapsid protein Abs emerged less consistently. Furthermore, we demonstrated the feasibility of finger-prick sampling for Ab detection against SARS-CoV-2 using these assays. In conclusion, the developed bridging assays reliably detect SARS-CoV-2 Abs in hospitalized and nonhospitalized patients and are therefore well suited to conduct seroprevalence studies.

Divergent SARS-CoV-2-specific T- and B-cell responses in severe but not mild COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2-specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4+ T-cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T-cell compartment, and indicative of vascular leakage. The observed disparate T- and B-cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients.