Monocyte Differentiation and Heterogeneity: Inter-Subset and Interindividual Differences.

The three subsets of human monocytes, classical, intermediate, and nonclassical, show phenotypic heterogeneity, particularly in their expression of CD14 and CD16. This has enabled researchers to delve into the functions of each subset in the steady state as well as in disease. Studies have revealed that monocyte heterogeneity is multi-dimensional. In addition, that their phenotype and function differ between subsets is well established. However, it is becoming evident that heterogeneity also exists within each subset, between health and disease (current or past) states, and even between individuals. This realisation casts long shadows, impacting how we identify and classify the subsets, the functions we assign to them, and how they are examined for alterations in disease. Perhaps the most fascinating is evidence that, even in relative health, interindividual differences in monocyte subsets exist. It is proposed that the individual's microenvironment could cause long-lasting or irreversible changes to monocyte precursors that echo to monocytes and through to their derived macrophages. Here, we will discuss the types of heterogeneity recognised in monocytes, the implications of these for monocyte research, and most importantly, the relevance of this heterogeneity for health and disease.

Characterization of anti-SARS-CoV-2 monoclonal antibodies focusing on antigen binding, neutralization, and FcγR activation via formation of immune complex.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Antibodies induced by SARS-CoV-2 infection or vaccination play pivotal roles in the body's defense against the virus; many monoclonal antibodies (mAbs) against SARS-CoV-2 have been cloned, and some neutralizing mAbs have been used as therapeutic drugs. In this study, we prepared an antibody panel consisting of 31 clones of anti-SARS-CoV-2 mAbs and analyzed and compared their biological activities. The mAbs used in this study were classified into different binding classes based on their binding epitopes and showed binding to the SARS-CoV-2 spike protein in different binding kinetics. A multiplex assay using the spike proteins of Alpha, Beta, Gamma, Delta, and Omicron variants clearly showed the different effects of variant mutations on the binding and neutralization activities of different binding classes of mAbs. In addition, we evaluated Fcγ receptor (FcγR) activation by immune complexes consisting of anti-SARS-CoV-2 mAb and SARS-CoV-2 pseudo-typed virus, and revealed differences in the FcγR activation properties among the binding classes of anti-SARS-CoV-2 mAbs. It has been reported that FcγR-mediated immune-cell activation by immune complexes is involved in the promotion of immunopathology of COVID-19; therefore, differences in the FcγR-activation properties of anti-SARS-CoV-2 mAbs are among the most important characteristics when considering the clinical impacts of anti-SARS-CoV-2 mAbs.

Delivery of a novel membrane-anchored Fc chimera enhances NK cell-mediated killing of tumor cells and persistently virus-infected cells.

Antibody-dependent cellular cytotoxicity (ADCC) is one of the most powerful mechanisms for Natural Killer (NK) cells to kill cancer cells or virus-infected cells. A novel chimeric protein (NA-Fc) was created, which when expressed in cells, positions an IgG Fc domain on the plasma membrane, mimicking the orientation of IgG bound to the cell surface. This NA-Fc chimera was tested with PM21-NK cells, produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Real time viability assays revealed higher PM21-NK killing of both ovarian and lung cancer cells expressing NA-Fc, which correlated with increased release of TNF-α and IFN-γ cytokines from NK cells and was dependent on CD16-Fc interactions. Lentivirus delivery of NA-Fc to target cells increased the rate of PM21-NK cell killing of A549 and H1299 lung, SKOV3 ovarian and A375 melanoma cancer cells. This NA-Fc-directed killing was extended to virus infected cells, where delivery of NA-Fc to lung cells that were persistently infected with Parainfluenza virus resulted in increased killing by PM21-NK cells. In contrast to its effect on PM21-NK cells, the NA-Fc molecule did not enhance complement mediated lysis of lung cancer cells. Our study lays the foundation for application of the novel NA-Fc chimera that could be delivered specifically to tumors during oncolytic virotherapy to mark target cells for ADCC by co-treatment with adoptive NK cells. This strategy would potentially eliminate the need to search for unique cancer specific antigens for development of new antibody therapeutics.

Asymptomatic COVID-19 Reinfection in a Pediatric Patient with Heterotaxy Syndrome.

We report an asymptomatic child with heterotaxy syndrome who had recurrent positive SARS-CoV-2 polymerase chain reaction testing. An aberrant lymphocyte population expressing CD19, CD16, and CD56 was identified; its phenotyping revealing atypical NK cells. This subset's role in protection from severe disease or in reinfection cannot be ascertained.

Fc-γR-dependent antibody effector functions are required for vaccine-mediated protection against antigen-shifted variants of SARS-CoV-2.

Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with antigenic changes in the spike protein are neutralized less efficiently by serum antibodies elicited by legacy vaccines against the ancestral Wuhan-1 virus. Nonetheless, these vaccines, including mRNA-1273 and BNT162b2, retained their ability to protect against severe disease and death, suggesting that other aspects of immunity control infection in the lung. Vaccine-elicited antibodies can bind Fc gamma receptors (FcγRs) and mediate effector functions against SARS-CoV-2 variants, and this property correlates with improved clinical coronavirus disease 2019 outcome. However, a causal relationship between Fc effector functions and vaccine-mediated protection against infection has not been established. Here, using passive and active immunization approaches in wild-type and FcγR-knockout mice, we determined the requirement for Fc effector functions to control SARS-CoV-2 infection. The antiviral activity of passively transferred immune serum was lost against multiple SARS-CoV-2 strains in mice lacking expression of activating FcγRs, especially murine FcγR III (CD16), or depleted of alveolar macrophages. After immunization with the pre-clinical mRNA-1273 vaccine, control of Omicron BA.5 infection in the respiratory tract also was lost in mice lacking FcγR III. Our passive and active immunization studies in mice suggest that Fc-FcγR engagement and alveolar macrophages are required for vaccine-induced antibody-mediated protection against infection by antigenically changed SARS-CoV-2 variants, including Omicron strains.

IgG antispike persistence and immunophenotype in children infected by SARS-CoV-2.

AIM: The immune status of children recovering from SARS-CoV-2 infection is not completely understood. We describe IgG antispike persistence in children infected during the first two pandemic waves. In addition, we compared with healthy controls their leukocyte populations and CD64 expression. METHODS: Cross-sectional study. Carried out from October 2021 to February 2022 in nonreinfected and nonvaccinated children with SARS-CoV-2 in 2020. The presence of antispike IgG was studied using chemiluminescent immunoassay. Leukocyte populations were analysed using flow cytometry and marked for CD45, CD4, CD8 and CD64. Statistical minor than 0.05 was considered significant. RESULTS: One hundred and eighty-three control and 77 patients were included. IgG antispike determinations were performed after a median of 501 days (262-464); 52 of 77 children were positive. Cases showed significantly higher percentages of monocytes, lymphocytes, CD8+ and CD4+ . In addition, CD64 expression was higher in monocytes and neutrophils. The presence of IgG antispike was accompanied by a higher percentage of CD64+ neutrophils. CONCLUSION: In our series, the SARS-CoV-2 IgG antispike protein was usually positive beyond 1 year after infection. Furthermore, leukocyte populations from cases differ from controls, with higher CD64 expression on neutrophils and monocytes. Prospective clinical observations are required to confirm the implications of these findings.

Heterogeneity in IgG-CD16 signaling in infectious disease outcomes.

In this review, we discuss how IgG antibodies can modulate inflammatory signaling during viral infections with a focus on CD16a-mediated functions. We describe the structural heterogeneity of IgG antibody ligands, including subclass and glycosylation that impact binding by and downstream activity of CD16a, as well as the heterogeneity of CD16a itself, including allele and expression density. While inflammation is a mechanism required for immune homeostasis and resolution of acute infections, we focus here on two infectious diseases that are driven by pathogenic inflammatory responses during infection. Specifically, we review and discuss the evolving body of literature showing that afucosylated IgG immune complex signaling through CD16a contributes to the overwhelming inflammatory response that is central to the pathogenesis of severe forms of dengue disease and coronavirus disease 2019 (COVID-19).

Dynamic Evaluation of Natural Killer Cells Subpopulations in COVID-19 Patients.

The aim of the study was to evaluate the dynamic changes of the total Natural Killer (NK) cells and different NK subpopulations according to their differentiated expression of CD16/CD56 in COVID-19 patients. Blood samples with EDTA were analyzed on day 1 (admission moment), day 5, and day 10 for the NK subtypes. At least 30,000 singlets were collected for each sample and white blood cells were gated in CD45/SSC and CD16/CD56 dot plots of fresh human blood. From the lymphocyte singlets, the NK cells subpopulations were analyzed based on the differentiated expression of surface markers and classified as follows: CD16-CD56+/++/CD16+CD56++/CD16+CD56+/CD16++CD56-. By examining the CD56 versus CD16 flow cytometry dot plots, we found four distinct NK sub-populations. These NK subtypes correspond to different NK phenotypes from secretory to cytolytic ones. There was no difference between total NK percentage of different disease forms. However, the total numbers decreased significantly both in survivors and non-survivors. Additionally, for the CD16-CD56+/++ phenotype, we observed different patterns, gradually decreasing in survivors and gradually increasing in those with fatal outcomes. Despite no difference in the proportion of the CD16-CD56++ NK cells in survivors vs. non-survivors, the main cytokine producers gradually decline during the study period in the survival group, underling the importance of adequate IFN production during the early stage of SARS-CoV-2 infection. Persistency in the circulation of CD56++ NK cells may have prognostic value in patients, with a fatal outcome. Total NK cells and the CD16+CD56+ NK subtypes exhibit significant decreasing trends across the moments for both survivors and non-survivors.

TIM3+ TRBV11-2 T cells and IFNγ signature in patrolling monocytes and CD16+ NK cells delineate MIS-C.

In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRß repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.

Cutting Edge: Hyperinflammatory Monocytes Expressing CD56 Abound in Severe COVID-19 Patients.

Proinflammatory monocytes play a preponderant role in the development of a cytokine storm causing fatal consequences in coronavirus disease 2019 (COVID-19) patients, highlighting the importance of analyzing in more detail monocyte distribution in these patients. In this study, we identified an atypical monocyte subpopulation expressing CD56 molecules that showed a low level of HLA-DR and high level of l-selectin. They released higher amounts of TNF-α and IL-6 and expressed genes associated with an excessive inflammatory process. Remarkably, the frequency of CD56+ monocytes inversely correlated with that of CD16+ monocytes and a high CD56+/CD16+monocyte ratio was associated with both disease severity and mortality, as well as with serum concentration of type I IFN, a factor able to induce the appearance of CD56+ monocytes. In conclusion, severe COVID-19 is characterized by the abundance of hyperinflammatory CD56+ monocytes, which could represent a novel marker with prognostic significance and, possibly, a therapeutic target for controlling the inflammatory process occurring during COVID-19.

FcγR-mediated SARS-CoV-2 infection of monocytes activates inflammation.

SARS-CoV-2 can cause acute respiratory distress and death in some patients1. Although severe COVID-19 is linked to substantial inflammation, how SARS-CoV-2 triggers inflammation is not clear2. Monocytes and macrophages are sentinel cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D, leading to inflammatory death (pyroptosis) and the release of potent inflammatory mediators3. Here we show that about 6% of blood monocytes of patients with COVID-19 are infected with SARS-CoV-2. Monocyte infection depends on the uptake of antibody-opsonized virus by Fcγ receptors. The plasma of vaccine recipients does not promote antibody-dependent monocyte infection. SARS-CoV-2 begins to replicate in monocytes, but infection is aborted, and infectious virus is not detected in the supernatants of cultures of infected monocytes. Instead, infected cells undergo pyroptosis mediated by activation of NLRP3 and AIM2 inflammasomes, caspase-1 and gasdermin D. Moreover, tissue-resident macrophages, but not infected epithelial and endothelial cells, from lung autopsies from patients with COVID-19 have activated inflammasomes. Taken together, these findings suggest that antibody-mediated SARS-CoV-2 uptake by monocytes and macrophages triggers inflammatory cell death that aborts the production of infectious virus but causes systemic inflammation that contributes to COVID-19 pathogenesis.

Fcγ-Receptor-Based Enzyme-Linked Immunosorbent Assays for Sensitive, Specific, and Persistent Detection of Anti-SARS-CoV-2 Nucleocapsid Protein IgG Antibodies in Human Sera.

Sensitive and specific serological tests are mandatory for epidemiological studies evaluating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevalence as well as coronavirus disease 2019 (COVID-19) morbidity and mortality rates. The accuracy of results is challenged by antibody waning after convalescence and by cross-reactivity induced by previous infections with other pathogens. By employing a patented platform technology based on capturing antigen-antibody complexes with a solid-phase-bound Fcγ receptor (FcγR) and truncated nucleocapsid protein as the antigen, two SARS-CoV-2 IgG enzyme-linked immunosorbent assays (ELISAs), featuring different serum and antigen dilutions, were developed. Validation was performed using a serum panel comprising 213 longitudinal samples from 35 COVID-19 patients and a negative-control panel consisting of 790 pre-COVID-19 samples from different regions of the world. While both assays show similar diagnostic sensitivities in the early convalescent phase, ELISA 2 (featuring a higher serum concentration) enables SARS-CoV-2 IgG antibody detection for a significantly longer time postinfection (≥15 months). Correspondingly, analytical sensitivity referenced to indirect immunofluorescence testing (IIFT) is significantly higher for ELISA 2 in samples with a titer of ≤1:640; for high-titer samples, a prozone effect is observed for ELISA 2. The specificities of both ELISAs were excellent not only for pre-COVID-19 serum samples from Europe, Asia, and South America but also for several challenging African sample panels. The SARS-CoV-2 IgG FcγR ELISAs, methodically combining antigen-antibody binding in solution and isotype-specific detection of immune complexes, are valuable tools for seroprevalence studies requiring the (long-term) detection of anti-SARS-CoV-2 IgG antibodies in populations with a challenging immunological background and/or in which spike-protein-based vaccine programs have been rolled out.

IgG-RBD Response Due to Inactivated SARS-CoV-2 Vaccine: Alteration in D-Dimer and Fibrinogen Concentrations, Association with Comorbidities and Adverse Effects.

OBJECTIVE: To examine the immunoglobulin G-receptor-binding domain (IgG-RBD) response and changes in fibrinogen and D-dimer concentrations in individuals with a past coronavirus infection and followed by CoronaVac. METHODS: The study consisted of a total of 116 participants. Blood samples were drawn from subjects 21-25 days after they received first and second doses of CoronaVac as well as from individuals with a past infection. Fibrinogen, D-dimer, and IgG-RBD concentrations were measured. RESULTS: The IgG concentrations of the vaccinated subjects were significantly higher (P < .001), fibrinogen levels were lower (P < .001), and D-dimer levels increased following the second vaccination compared with the first vaccination (P = .083). No difference was obtained in IgG-RBD between vaccinated and previously infected individuals (P = .063). The differences in fibrinogen and D-dimer were statistically nonsignificant between both groups. CONCLUSION: The CoronaVac vaccine appears to be safe and effective. It is essential for individuals to take personal protective measures, such as using masks and distancing.

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.

CD66b-CD64dimCD115- cells in the human bone marrow represent neutrophil-committed progenitors.

Here we report the identification of human CD66b-CD64dimCD115- neutrophil-committed progenitor cells (NCPs) within the SSCloCD45dimCD34+ and CD34dim/- subsets in the bone marrow. NCPs were either CD45RA+ or CD45RA-, and in vitro experiments showed that CD45RA acquisition was not mandatory for their maturation process. NCPs exclusively generated human CD66b+ neutrophils in both in vitro differentiation and in vivo adoptive transfer experiments. Single-cell RNA-sequencing analysis indicated NCPs fell into four clusters, characterized by different maturation stages and distributed along two differentiation routes. One of the clusters was characterized by an interferon-stimulated gene signature, consistent with the reported expansion of peripheral mature neutrophil subsets that express interferon-stimulated genes in diseased individuals. Finally, comparison of transcriptomic and phenotypic profiles indicated NCPs represented earlier neutrophil precursors than the previously described early neutrophil progenitors (eNePs), proNeus and COVID-19 proNeus. Altogether, our data shed light on the very early phases of neutrophil ontogeny.

Signaling Through FcγRIIA and the C5a-C5aR Pathway Mediate Platelet Hyperactivation in COVID-19.

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.

Inflammasome activation in infected macrophages drives COVID-19 pathology.

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA and a sustained interferon (IFN) response, all of which are recapitulated and required for pathology in the SARS-CoV-2-infected MISTRG6-hACE2 humanized mouse model of COVID-19, which has a human immune system1-20. Blocking either viral replication with remdesivir21-23 or the downstream IFN-stimulated cascade with anti-IFNAR2 antibodies in vivo in the chronic stages of disease attenuates the overactive immune inflammatory response, especially inflammatory macrophages. Here we show that SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release interleukin 1 (IL-1) and IL-18, and undergo pyroptosis, thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and the accompanying inflammatory response are necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Notably, this blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 through the production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

PIMS-TS immunophenotype: description and comparison with healthy children, Kawasaki disease and severe viral and bacterial infections.

BACKGROUND: A new clinical syndrome named Paediatric Inflammatory Multisystem Syndrome Temporally Associated with SARS-CoV-2 (PIMS-TS) has been described. This new disease is a leading cause of hospital and paediatric intensive care unit (PICU). It has been related to immunity dysregulation. METHODS: Prospective-retrospective observational study to describe the innate cell signature and immunophenotype of children admitted to PICU because of PIMS-TS (from March 2020 to September 2020). The immunophenotype was done through the expression analysis of these proteins of mononuclear cells: CD64, CD18, CD11a and CD11b. They were compared with previous healthy controls and children admitted to PICU because of bacterial infection, viral infection and Kawasaki disease (KD). Two hundred and forty-seven children were studied: 183 healthy controls, 25 viral infections, 20 bacterial infections, 6 KD and 13 PIMS-TS. RESULTS: PIMT-TS showed the lowest percentage of lymphocytes and monocytes with higher relative numbers of CD4+ (p = .000). Monocytes and neutrophils in PIMS-TS showed higher levels of CD64 expression (p = .000). Also, CD11a and CD11b were highly expressed (p =,000). CONCLUSION: We observed a differential cell innate signature in PIMS-TS. These findings are consistent with a proinflammatory status (CD64 elevated expression) and lymphocyte trafficking to tissues (CD11a and CD11b). More studies should be carried out to confirm our results.

The deglycosylated form of 1E12 inhibits platelet activation and prothrombotic effects induced by VITT antibodies.

In order to improve the safety of COVID-19 vaccines, there is an urgent need to unravel the pathogenesis of vaccineinduced immune thrombotic thrombocytopenia (VITT), a severe complication of recombinant adenoviral vector vaccines used to prevent COVID-19, and likely due to anti-platelet factor 4 (PF4) IgG antibodies. In this study, we demonstrated that 1E12, a chimeric anti-PF4 antibody with a human Fc fragment, fully mimics the effects of human VITT antibodies, as it activates platelets to a similar level in the presence of platelet factor 4 (PF4). Incubated with neutrophils, platelets and PF4, 1E12 also strongly induces NETosis, and in a microfluidic model of whole blood thrombosis, it triggers the formation of large platelet/leukocyte thrombi containing fibrin(ogen). In addition, a deglycosylated form of 1E12 (DG-1E12), which still binds PF4 but no longer interacts with Fcγ receptors, inhibits platelet, granulocyte and clotting activation induced by human anti-PF4 VITT antibodies. This strongly supports that 1E12 and VITT antibodies recognize overlapping epitopes on PF4. In conclusion, 1E12 is a potentially important tool to study the pathophysiology of VITT, and for establishing mouse models. On the other hand, DG-1E12 may help the development of a new drug that specifically neutralizes the pathogenic effect of autoimmune anti-PF4 antibodies, such as those associated with VITT.

Multiple Routes of Antibody-Dependent Enhancement of SARS-CoV-2 Infection.

Antibody-dependent enhancement (ADE) of infection is generally known for many viruses. A potential risk of ADE in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has also been discussed since the beginning of the coronavirus disease 2019 (COVID-19) pandemic; however, clinical evidence of the presence of antibodies with ADE potential is limited. Here, we show that ADE antibodies are produced by SARS-CoV-2 infection and the ADE process can be mediated by at least two different host factors, Fcγ receptor (FcγR) and complement component C1q. Of 89 serum samples collected from acute or convalescent COVID-19 patients, 62.9% were found to be positive for SARS-CoV-2-specific IgG. FcγR- and/or C1q-mediated ADE were detected in 50% of the IgG-positive sera, whereas most of them showed neutralizing activity in the absence of FcγR and C1q. Importantly, ADE antibodies were found in 41.4% of the acute COVID-19 patients. Neutralizing activity was also detected in most of the IgG-positive sera, but it was counteracted by ADE in subneutralizing conditions in the presence of FcγR or C1q. Although the clinical importance of ADE needs to be further investigated with larger numbers of COVID-19 patient samples, our data suggest that SARS-CoV-2 utilizes multiple mechanisms of ADE. C1q-mediated ADE may particularly have a clinical impact since C1q is present at high concentrations in plasma and its receptors are ubiquitously expressed on the surfaces of many types of cells, including respiratory epithelial cells, which SARS-CoV-2 primarily infects. IMPORTANCE Potential risks of antibody-dependent enhancement (ADE) in the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been discussed and the proposed mechanism mostly depends on the Fc gamma receptor (FcγR). However, since FcγRs are exclusively expressed on immune cells, which are not primary targets of SARS-CoV-2, the clinical importance of ADE of SARS-CoV-2 infection remains controversial. Our study demonstrates that SARS-CoV-2 infection induces antibodies that increase SARS-CoV-2 infection through another ADE mechanism in which complement component C1q mediates the enhancement. Although neutralizing activity was also detected in the serum samples, it was counteracted by ADE in the presence of FcγR or C1q. Considering the ubiquity of C1q and its cellular receptors, C1q-mediated ADE may more likely occur in respiratory epithelial cells, which SARS-CoV-2 primarily infects. Our data highlight the importance of careful monitoring of the antibody properties in COVID-19 convalescent and vaccinated individuals.