Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

Paediatric inflammatory multisystem syndrome in Canada: population-based surveillance and role of SARS-CoV-2 linkage.

BACKGROUND: Paediatric inflammatory multisystem syndrome (PIMS) is a rare condition temporally associated with SARS-CoV-2 infection. Using national surveillance data, we compare presenting features and outcomes among children hospitalized with PIMS by SARS-CoV-2 linkage, and identify risk factors for intensive care (ICU). METHODS: Cases were reported to the Canadian Paediatric Surveillance Program by a network of >2800 pediatricians between March 2020 and May 2021. Patients with positive versus negative SARS-CoV-2 linkages were compared, with positive linkage defined as any positive molecular or serologic test or close contact with confirmed COVID-19. ICU risk factors were identified with multivariable modified Poisson regression. RESULTS: We identified 406 children hospitalized with PIMS, including 49.8% with positive SARS-CoV-2 linkages, 26.1% with negative linkages, and 24.1% with unknown linkages. The median age was 5.4 years (IQR 2.5-9.8), 60% were male, and 83% had no comorbidities. Compared to cases with negative linkages, children with positive linkages experienced more cardiac involvement (58.8% vs. 37.4%; p < 0.001), gastrointestinal symptoms (88.6% vs. 63.2%; p < 0.001), and shock (60.9% vs. 16.0%; p < 0.001). Children aged ≥6 years and those with positive linkages were more likely to require ICU. CONCLUSIONS: Although rare, 30% of PIMS hospitalizations required ICU or respiratory/hemodynamic support, particularly those with positive SARS-CoV-2 linkages. IMPACT: We describe 406 children hospitalized with paediatric inflammatory multisystem syndrome (PIMS) using nationwide surveillance data, the largest study of PIMS in Canada to date. Our surveillance case definition of PIMS did not require a history of SARS-CoV-2 exposure, and we therefore describe associations of SARS-CoV-2 linkages on clinical features and outcomes of children with PIMS. Children with positive SARS-CoV-2 linkages were older, had more gastrointestinal and cardiac involvement, and hyperinflammatory laboratory picture. Although PIMS is rare, one-third required admission to intensive care, with the greatest risk amongst those aged ≥6 years and those with a SARS-CoV-2 linkage.

Deciphering Fc-effector functions against SARS-CoV-2.

Major efforts were deployed to study the antibody response against SARS-CoV-2. Antibodies neutralizing SARS-CoV-2 have been extensively studied in the context of infections, vaccinations, and breakthrough infections. Antibodies, however, are pleiotropic proteins that have many functions in addition to neutralization. These include Fc-effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Although important to combat viral infections, these Fc-effector functions were less studied in the context of SARS-CoV-2 compared with binding and neutralization. This is partly due to the difficulty in developing reliable assays to measure Fc-effector functions compared to antibody binding and neutralization. Multiple assays have now been developed and can be used to measure different Fc-effector functions. Here, we review these assays and what is known regarding anti-SARS-CoV-2 Fc-effector functions. Overall, this review summarizes and updates our current state of knowledge regarding anti-SARS-CoV-2 Fc-effector functions.

NTB-A and 2B4 Natural Killer Cell Receptors Modulate the Capacity of a Cocktail of Non-Neutralizing Antibodies and a Small CD4-Mimetic to Eliminate HIV-1-Infected Cells by Antibody-Dependent Cellular Cytotoxicity.

Natural Killer (NK) cells have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC). NK cell activation is tightly regulated by the engagement of its inhibitory and activating receptors. The activating receptor CD16 drives ADCC upon binding to the Fc portion of antibodies; NK cell activation is further sustained by the co-engagement of activating receptors NTB-A and 2B4. During HIV-1 infection, Nef and Vpu accessory proteins contribute to ADCC escape by downregulating the ligands of NTB-A and 2B4. HIV-1 also evades ADCC by keeping its envelope glycoproteins (Env) in a "closed" conformation which effectively masks epitopes recognized by non-neutralizing antibodies (nnAbs) which are abundant in the plasma of people living with HIV. To achieve this, the virus uses its accessory proteins Nef and Vpu to downregulate the CD4 receptor, which otherwise interacts with Env and exposes the epitopes recognized by nnAbs. Small CD4-mimetic compounds (CD4mc) have the capacity to expose these epitopes, thus sensitizing infected cells to ADCC. Given the central role of NK cell co-activating receptors NTB-A and 2B4 in Fc-effector functions, we studied their contribution to CD4mc-mediated ADCC. Despite the fact that their ligands are partially downregulated by HIV-1, we found that both co-activating receptors significantly contribute to CD4mc sensitization of HIV-1-infected cells to ADCC.

Intra-Host Evolution Analyses in an Immunosuppressed Patient Supports SARS-CoV-2 Viral Reservoir Hypothesis.

Throughout the SARS-CoV-2 pandemic, several variants of concern (VOCs) have been identified, many of which share recurrent mutations in the spike glycoprotein's receptor-binding domain (RBD). This region coincides with known epitopes and can therefore have an impact on immune escape. Protracted infections in immunosuppressed patients have been hypothesized to lead to an enrichment of such mutations and therefore drive evolution towards VOCs. Here, we present the case of an immunosuppressed patient that developed distinct populations with immune escape mutations throughout the course of their infection. Notably, by investigating the co-occurrence of substitutions on individual sequencing reads in the RBD, we found quasispecies harboring mutations that confer resistance to known monoclonal antibodies (mAbs) such as S:E484K and S:E484A. These mutations were acquired without the patient being treated with mAbs nor convalescent sera and without them developing a detectable immune response to the virus. We also provide additional evidence for a viral reservoir based on intra-host phylogenetics, which led to a viral substrain that evolved elsewhere in the patient's body, colonizing their upper respiratory tract (URT). The presence of SARS-CoV-2 viral reservoirs can shed light on protracted infections interspersed with periods where the virus is undetectable, and potential explanations for long-COVID cases.

Three families of CD4-induced antibodies are associated with the capacity of plasma from people living with HIV to mediate ADCC in presence of CD4-mimetics.

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation which occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in presence of CD4mc.

The combination of three CD4-induced antibodies targeting highly conserved Env regions with a small CD4-mimetic achieves potent ADCC activity.

The majority of naturally-elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs), because they are unable to recognize the Env timer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by Antibody-Dependent Cellular Cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly-neutralizing antibodies and even showed activity against HIV-1 infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies.

The Fc-effector function of COVID-19 convalescent plasma contributes to SARS-CoV-2 treatment efficacy in mice.

COVID-19 convalescent plasmas (CCPs) are chosen for plasma therapy based on neutralizing titers and anti-Spike immunoglobulin levels. However, CCP characteristics that promote SARS-CoV-2 control are complex and incompletely defined. Using an in vivo imaging approach, we demonstrate that CCPs with low neutralizing (ID50 ≤ 1:250), but moderate to high Fc-effector activity, in contrast to those with poor Fc function, delay mortality and/or improve survival of SARS-CoV-2-challenged K18-hACE2 mice. The impact of innate immune cells on CCP efficacy depended on their residual neutralizing activity. Fractionation of a selected CCP revealed that IgG and Ig(M + A) were required during therapy, but the IgG fraction alone sufficed during prophylaxis. Finally, despite reduced neutralization, ancestral SARS-CoV-2-elicited CCPs significantly delayed Delta and Beta-induced mortality suggesting that Fc-effector functions contribute to immunity against VOCs. Thus, Fc activity of CCPs provide a second line of defense when neutralization is compromised and can serve as an important criterion for CCP selection.

A Recent SARS-CoV-2 Infection Enhances Antibody-Dependent Cellular Cytotoxicity against Several Omicron Subvariants following a Fourth mRNA Vaccine Dose.

Since the beginning of the SARS-CoV-2 pandemic, several variants of concern (VOCs), such as the Alpha, Beta, Gamma, Delta and Omicron variants, have arisen and spread worldwide. Today, the predominant circulating subvariants are sublineages of the Omicron variant, which have more than 30 mutations in their Spike glycoprotein compared to the ancestral strain. The Omicron subvariants were significantly less recognized and neutralized by antibodies from vaccinated individuals. This resulted in a surge in the number of infections, and booster shots were recommended to improve responses against these variants. While most studies mainly measured the neutralizing activity against variants, we and others previously reported that Fc-effector functions, including antibody-dependent cellular cytotoxicity (ADCC), play an important role in humoral responses against SARS-CoV-2. In this study, we analyzed Spike recognition and ADCC activity against several Omicron subvariants by generating cell lines expressing different Omicron subvariant Spikes. We tested these responses in a cohort of donors, who were recently infected or not, before and after a fourth dose of mRNA vaccine. We showed that ADCC activity is less affected than neutralization by the antigenic shift of the tested Omicron subvariant Spikes. Moreover, we found that individuals with a history of recent infection have higher antibody binding and ADCC activity against all Omicron subvariants than people who were not recently infected. With an increase in the number of reinfections, this study helps better understand Fc-effector responses in the context of hybrid immunity.

Elevated binding and functional antibody responses to SARS-CoV-2 in infants versus mothers.

Infant antibody responses to viral infection can differ from those in adults. However, data on the specificity and function of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in infants, and direct comparisons between infants and adults are limited. We characterized antibody binding and functionality in convalescent plasma from postpartum women and their infants infected with SARS-CoV-2 from a vaccine-naïve prospective cohort in Nairobi, Kenya. Antibody titers against SARS-CoV-2 Spike, receptor binding domain and N-terminal domain, and Spike-expressing cell-surface staining levels were significantly higher in infants than in mothers. Plasma antibodies from mothers and infants bound to similar regions of the Spike S2 subunit, including the fusion peptide (FP) and stem helix-heptad repeat 2. However, infants displayed higher antibody levels and more consistent antibody escape pathways in the FP region compared to mothers. Finally, infants had significantly higher levels of antibody-dependent cellular cytotoxicity (ADCC), though, surprisingly, neutralization titers between infants and mothers were similar. These results suggest infants develop distinct SARS-CoV-2 binding and functional antibody repertoires and reveal age-related differences in humoral immunity to SARS-CoV-2 infection that could be relevant to protection and COVID-19 disease outcomes.

Small CD4 mimetics sensitize HIV-1-infected macrophages to antibody-dependent cellular cytotoxicity.

HIV-1 envelope (Env) conformation determines the susceptibility of infected CD4+ T cells to antibody-dependent cellular cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more "open" conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu, and Env. Limited data exist, however, of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu, and Env are all required to efficiently downregulate CD4 on infected CD4+ T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared with CD4+ T cells. However, treatment of infected macrophages with small CD4 mimetics exposes vulnerable CD4-induced Env epitopes and sensitizes them to ADCC.

Humoral Responses Elicited after a Fifth Dose of SARS-CoV-2 mRNA Bivalent Vaccine.

While an important part of the world's population is vaccinated against SARS-CoV-2, new variants continue to emerge. We observe that even after a fifth dose of the mRNA bivalent vaccine, most vaccinated individuals have antibodies that poorly neutralize several Omicron subvariants, including BQ.1.1, XBB, XBB.1.5, FD.1.1, and CH.1.1. However, Fc-effector functions remain strong and stable over time against new variants, which may partially explain why vaccines continue to be effective. We also observe that donors who have been recently infected have stronger antibody functional activities, including neutralization and Fc-effector functions, supporting the observations that hybrid immunity leads to better humoral responses.

Elevated binding and functional antibody responses to SARS-CoV-2 in infants versus mothers.

Infant antibody responses to viral infection can differ from those in adults. However, data on the specificity and function of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in infants, and direct comparisons between infants and adults are limited. Here, we characterize antibody binding and functionality against Wuhan-Hu-1 (B lineage) strain SARS-CoV-2 in convalescent plasma from 36 postpartum women and 14 of their infants infected with SARS-CoV-2 from a vaccine-naïve prospective cohort in Nairobi, Kenya. We find significantly higher antibody titers against SARS-CoV-2 Spike, receptor binding domain and N-terminal domain, and Spike-expressing cell-surface staining levels in infants versus mothers. Plasma antibodies from mothers and infants bind to similar regions of the Spike S2 subunit, including the fusion peptide (FP) and stem helix-heptad repeat 2. However, infants display higher antibody levels and more consistent antibody escape pathways in the FP region compared to mothers. Finally, infants have significantly higher levels of antibody-dependent cellular cytotoxicity (ADCC), though, surprisingly, Spike pseudovirus neutralization titers between infants and mothers are similar. These results suggest infants develop distinct SARS-CoV-2 binding and functional antibody activities and reveal age-related differences in humoral immunity to SARS-CoV-2 infection that could be relevant to protection and COVID-19 disease outcomes.

Molecular basis for antiviral activity of two pediatric neutralizing antibodies targeting SARS-CoV-2 Spike RBD.

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV-2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.

Resource use and disease severity of children hospitalized for COVID-19 versus multisystem inflammatory syndrome in children (MIS-C) in Canada.

Background: Direct comparisons of paediatric hospitalizations for acute coronavirus disease 2019 (COVID-19) and multisystem inflammatory syndrome in children (MIS-C) can inform health system planning. We describe the absolute and relative hospital burden of acute paediatric COVID-19 and MIS-C in Canada. Methods: This national prospective study was conducted via the Canadian Paediatric Surveillance Program from March 2020-May 2021. Children younger than 18 years old and hospitalized for acute COVID-19 or MIS-C were included in the analysis. Outcomes included supplemental oxygen (low-flow oxygen or high-flow nasal cannula), ventilation (non-invasive or conventional mechanical), vasopressors, paediatric intensive care unit (PICU) admission, or death. Adjusted risk differences (aRD) and 95% confidence intervals (CI) were calculated to identify factors associated with each diagnosis. Results: Overall, we identified 330 children hospitalized for acute COVID-19 (including five deaths) and 208 hospitalized for MIS-C (including zero deaths); PICU admission was required for 49.5% of MIS-C hospitalizations versus 18.2% of acute COVID-19 hospitalizations (aRD 20.3; 95% CI, 9.9-30.8). Resource use differed by age, with children younger than one year hospitalized more often for acute COVID-19 (aRD 43.4% versus MIS-C; 95% CI, 37.7-49.1) and more children 5-11 years hospitalized for MIS-C (aRD 38.9% vs. acute COVID-19; 95% CI, 31.0-46.9). Conclusion: While there were more hospitalizations and deaths from acute paediatric COVID-19, MIS-C cases were more severe, requiring more intensive care and vasopressor support. Our findings suggest that both acute COVID-19 and MIS-C should be considered when assessing the overall burden of severe acute respiratory syndrome coronavirus 2 in hospitalized children.

SARS-CoV-2 Accessory Protein ORF8 Decreases Antibody-Dependent Cellular Cytotoxicity.

Viruses use many different strategies to evade host immune responses. In the case of SARS-CoV-2, its Spike mutates rapidly to escape from neutralizing antibodies. In addition to this strategy, ORF8, a small accessory protein encoded by SARS-CoV-2, helps immune evasion by reducing the susceptibility of SARS-CoV-2-infected cells to the cytotoxic CD8+ T cell response. Interestingly, among all accessory proteins, ORF8 is rapidly evolving and a deletion in this protein has been linked to milder disease. Here, we studied the effect of ORF8 on peripheral blood mononuclear cells (PBMC). Specifically, we found that ORF8 can bind monocytes as well as NK cells. Strikingly, ORF8 binds CD16a (FcγRIIIA) with nanomolar affinity and decreases the overall level of CD16 at the surface of monocytes and, to a lesser extent, NK cells. This decrease significantly reduces the capacity of PBMCs and particularly monocytes to mediate antibody-dependent cellular cytotoxicity (ADCC). Overall, our data identifies a new immune-evasion activity used by SARS-CoV-2 to escape humoral responses.

Temperature Influences the Interaction between SARS-CoV-2 Spike from Omicron Subvariants and Human ACE2.

SARS-CoV-2 continues to infect millions of people worldwide. The subvariants arising from the variant-of-concern (VOC) Omicron include BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4, and BA.5. All possess multiple mutations in their Spike glycoprotein, notably in its immunogenic receptor-binding domain (RBD), and present enhanced viral transmission. The highly mutated Spike glycoproteins from these subvariants present different degrees of resistance to recognition and cross-neutralisation by plasma from previously infected and/or vaccinated individuals. We have recently shown that the temperature affects the interaction between the Spike and its receptor, the angiotensin converting enzyme 2 (ACE2). The affinity of RBD for ACE2 is significantly increased at lower temperatures. However, whether this is also observed with the Spike of Omicron and sub-lineages is not known. Here we show that, similar to other variants, Spikes from Omicron sub-lineages bind better the ACE2 receptor at lower temperatures. Whether this translates into enhanced transmission during the fall and winter seasons remains to be determined.

Association between SARS-CoV-2 RNAemia and dysregulated immune response in acutely ill hospitalized COVID-19 patients.

Severe/critical COVID-19 is associated with immune dysregulation and plasmatic SARS-CoV-2 detection (i.e. RNAemia). We detailed the association of SARS-CoV-2 RNAemia with immune responses in COVID-19 patients at the end of the first week of disease. We enrolled patients hospitalized in acute phase of ascertained SARS-CoV-2 pneumonia, and evaluated SARS-CoV-2 RNAemia, plasmatic cytokines, activated/pro-cytolytic T-cells phenotypes, SARS-CoV-2-specific cytokine-producing T-cells (IL-2, IFN-γ, TNF-α, IL-4, IL-17A), simultaneous Th1-cytokines production (polyfunctionality) and amount (iMFI). The humoral responses were assessed with anti-S1/S2 IgG, anti-RBD total-Ig, IgM, IgA, IgG1 and IgG3, neutralization and antibody-dependent cellular cytotoxicity (ADCC). Out of 54 patients, 27 had detectable viremia (viremic). Albeit comparable age and co-morbidities, viremic more frequently required ventilatory support, with a trend to higher death. Viremic displayed higher pro-inflammatory cytokines (IFN-α, IL-6), lower activated T-cells (HLA-DR+CD38+), lower functional SARS-CoV-2-specific T-cells (IFN-γ+CD4+, TNF-α+CD8+, IL-4+CD8+, IL-2+TNF-α+CD4+, and IL-2+TNF-α+CD4+ iMFI) and SARS-CoV-2-specific Abs (anti-S IgG, anti-RBD total-Ig, IgM, IgG1, IgG3; ID50, %ADCC). These data suggest a link between SARS-CoV-2 RNAemia at the end of the first stage of disease and immune dysregulation. Whether high ab initium viral burden and/or intrinsic host factors contribute to immune dysregulation in severe COVID-19 remains to be elucidated, to further inform strategies of targeted therapeutic interventions.

Humoral immune responses against SARS-CoV-2 Spike variants after mRNA vaccination in solid organ transplant recipients.

Although SARS-CoV-2 mRNA vaccination has been shown to be safe and effective in the general population, immunocompromised solid organ transplant recipients (SOTRs) were reported to have impaired immune responses after one or two doses of vaccine. In this study, we examined humoral responses induced after the second and the third dose of mRNA vaccine in different SOTR (kidney, liver, lung, and heart). Compared to a cohort of SARS-CoV-2 naïve immunocompetent health care workers (HCWs), the second dose induced weak humoral responses in SOTRs, except for the liver recipients. The third dose boosted these responses but they did not reach the same level as in HCW. Interestingly, although the neutralizing activity against Delta and Omicron variants remained very low after the third dose, Fc-mediated effector functions in SOTR reached similar levels as in the HCW cohort. Whether these responses will suffice to protect SOTR from severe outcome remains to be determined.