SARS-CoV-2 Antibodies Mediate Complement and Cellular Driven Inflammation

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to constitute a serious public health threat worldwide. Protective antibody-mediated viral neutralization in response to SARS-CoV-2 infection has been firmly characterized. Where the effects of the antibody response are generally considered to be beneficial, an important biological question regarding potential negative outcomes of a SARS-CoV-2 antibody response has yet to be answered. We determined the distribution of IgG subclasses and complement activation levels in plasma from convalescent individuals using in-house developed ELISAs. The IgG response towards SARS-CoV-2 receptor-binding domain (RBD) after natural infection appeared to be mainly driven by IgG1 and IgG3 subclasses, which are the main ligands for C1q mediated classical complement pathway activation. The deposition of the complement components C4b, C3bc, and TCC as a consequence of SARS-CoV-2 specific antibodies were depending primarily on the SARS-CoV-2 RBD and significantly correlated with both IgG levels and disease severity, indicating that individuals with high levels of IgG and/or severe disease, might have a more prominent complement activation during viral infection. Finally, freshly isolated monocytes and a monocyte cell line (THP-1) were used to address the cellular mediated inflammatory response as a consequence of Fc-gamma receptor engagement by SARS-CoV-2 specific antibodies. Monocytic Fc gamma receptor charging resulted in a significant rise in the secretion of the pro-inflammatory cytokine TNF-α. Our results indicate that SARS-CoV-2 antibodies might drive significant inflammatory responses through the classical complement pathway and via cellular immune-complex activation that could have negative consequences during COVID-19 disease. We found that increased classical complement activation was highly associated to COVID-19 disease severity. The combination of antibody-mediated complement activation and subsequent cellular priming could constitute a significant risk of exacerbating COVID-19 severity.

Fc-gamma receptor expression and cytokine response to intravenous human immunoglobulin in mothers and neonates

ObjectiveThe use of intravenous human immunoglobulin (IVIg) as adjuvant therapy for sepsis has been shown efficacious in adults, but its use in pregnant women and newborns is controversial. Fc gamma receptors (FcγRs) support the ability of IVIg to stimulate the synthesis of inflammatory mediators and promote phagocytosis by leukocytes, however, the FcγRs expression is differential between adults and neonates. We aimed to explore the effect of IVIg in monocytes and neutrophils from mother and neonates in whole blood cultures.Study designWhole blood from adults, maternal, and neonates were incubated with LPS and/or IVIg. After 0, 24, and 48 hours, we measured the expression of FcγRs (CD16, CD32, and CD64) and bacterial phagocytosis by monocytes and neutrophils. Also, the concentration of pro-inflammatory cytokines/chemokines was determined.ResultsFcγRs expression is quite similar among groups, and the LPS or IVIg challenge did not change the FcγRs expression on monocytes and neutrophils. Also, the LPS or IVIg challenge did not modify phagocytosis capacity in any group. However, IVIg induces a higher IL-8 response in neonates than in adults.ConclusionOur results suggest that the IL-8 response to IVIg in whole blood from neonates is not dependent on differential FcγR expression.Key messagesIVIg challenge in neonates or adults does not induce FcγR change expression on monocytes or neutrophilsIVIg induces higher IL-8 response in neonates than in adults

Humoral immune responses against seasonal coronaviruses predict efficiency of SARS-CoV-2 spike targeting, FcγR activation, and corresponding COVID-19 disease severity

Despite SARS-CoV-2 being a "novel" coronavirus, several studies suggest that detection of anti-spike IgG early in infection may be attributable to the amplification of humoral memory responses against seasonal hCoVs in severe COVID-19 patients. In this study, we examined this concept by characterizing anti-spike IgG from a cohort of non-hospitalized convalescent individuals with a spectrum of COVID-19 severity. We observed that anti-spike IgG levels positively correlated with disease severity, higher IgG cross-reactivity against betacoronaviruses (SARS-CoV-1 and OC43), and higher levels of proinflammatory Fc gamma receptor 2a and 3a (FcγR2a & FcγR3a) activation. In examining the levels of IgG targeting betacoronavirus conserved and immunodominant epitopes versus disease severity, we observed a positive correlation with the levels of IgG targeting the conserved S2'FP region, and an inverse correlation with two conserved epitopes around the heptad repeat (HR) 2 region. In comparing the levels of IgG targeting non-conserved epitopes, we observed that only one of three non-conserved immunodominant epitopes correlated with disease severity. Notably, the levels of IgG targeting the receptor binding domain (RBD) were inversely correlated with severity. Importantly, targeting of the RBD and HR2 regions have both been shown to mediate SARS-CoV-2 neutralization. These findings show that, aside from antibody (Ab) targeting of the RBD region, humoral memory responses against seasonal betacoronaviruses are potentially an important factor in dictating COVID-19 severity, with anti-HR2-dominant Ab profiles representing protective memory responses, while an anti-S2'FP dominant Ab profiles indicate deleterious recall responses. Though these profiles are masked in whole antigen profiling, these analyses suggest that distinct Ab memory responses are detectable with epitope targeting analysis. These findings have important implications for predicting severity of SARS-CoV-2 infections (primary and reinfections), and may predict vaccine efficacy in subpopulations with different dominant antibody epitope profiles.

Functional Fc Gamma Receptor Gene Polymorphisms and Long-Term Kidney Allograft Survival

The functional Fc gamma receptor (FcγR) IIIA polymorphism FCGR3A-V/F158 was earlier suggested to determine the potential of donor-specific HLA antibodies to trigger microcirculation inflammation, a key lesion of antibody-mediated renal allograft rejection. Associations with long-term transplant outcomes, however, have not been evaluated to date. To clarify the impact of FCGR3A-V/F158 polymorphism on kidney transplant survival, we genotyped a cohort of 1,940 recipient/donor pairs. Analyzing 10-year death-censored allograft survival, we found no significant differences in relation to FCGR3A-V/F158. There was also no independent survival effect in a multivariable Cox model. Similarly, functional polymorphisms in two other activating FcγR, FCGR2A-H/R131 (FcγRIIA) and FCGR3B-NA1/NA2 (FcγRIIIB), were not associated with outcome. There were also no significant survival differences among patient subgroups at increased risk of rejection-related injury, such as pre-sensitized recipients (> 0% panel reactivity; n = 438) or recipients treated for rejection within the first year after transplantation (n = 229). Our study results suggest that the earlier shown association of FcγR polymorphism with microcirculation inflammation may not be strong enough to exert a meaningful effect on graft survival.

Neutrophils dominate in opsonic phagocytosis of P. falciparum blood-stage merozoites and protect against febrile malaria

Antibody-mediated opsonic phagocytosis (OP) of Plasmodium falciparum blood-stage merozoites has been associated with protection against malaria. However, the precise contribution of different peripheral blood phagocytes in the OP mechanism remains unknown. Here, we developed an in vitro OP assay using peripheral blood leukocytes that allowed us to quantify the contribution of each phagocytic cell type in the OP of merozoites. We found that CD14 + +CD16− monocytes were the dominant phagocytic cells at very low antibody levels and Fc gamma receptor (FcγR) IIA plays a key role. At higher antibody levels however, neutrophils were the main phagocytes in the OP of merozoites with FcγRIIIB acting synergistically with FcγRIIA in the process. We found that OP activity by neutrophils was strongly associated with protection against febrile malaria in longitudinal cohort studies performed in Ghana and India. Our results demonstrate that peripheral blood neutrophils are the main phagocytes of P. falciparum blood-stage merozoites.

AB0756 IMMUNE-MEDIATED BASIS FOR A PHASE 2A CLINICAL STUDY COMPARING RILZABRUTINIB VS GLUCOCORTICOIDS IN RITUXIMAB-REFRACTORY PATIENTS WITH IGG4-RELATED DISEASE

Background:IgG4-related disease (IgG4-RD) is an immune-mediated disorder causing fibro-inflammatory lesions. Although the cause remains unknown, it may be driven by interactions between B lymphocytes and CD4+ cytotoxic and regulatory T cells and is characterized by an increase in short-lived plasmablasts, circulating antibodies, and macrophages. Standard therapy mainly includes glucocorticoids (GC), limited by toxicity with long-term use (> 6 mo), and to a lesser extent, immunosuppressives (eg, rituximab). Bruton tyrosine kinase (BTK) plays an important role in the activation of multiple immune effector cells such as B cells, mast cells, eosinophils, basophils, monocytes/macrophages, and neutrophils. Dysregulation of the activation of these immune cells results in autoimmune inflammation, tissue damage, and development of fibrosis. Rilzabrutinib is a highly selective oral BTK inhibitor that targets multiple pathways of innate and adaptive immunity (with direct effects on B-cell and FcR pathways) and has the potential to inhibit antigen presentation to autoreactive T cells.Objectives:To provide the biological rationale for rilzabrutinib in IgG4-RD.Methods:Rilzabrutinib has been evaluated in biochemical, in vitro studies, and in vivo models of inflammatory diseases. Additional support is provided by the phase 2 trial for oral rilzabrutinib in patients with pemphigus vulgaris and the phase 2 trial for oral rilzabrutinib in patients with immune thrombocytopenia (ITP).Results:Rilzabrutinib inhibited the activity of BTK and B-cell receptor in B cells (IC50 5-123 nM) and Fc gamma receptor in IgG/Fc gamma receptor-stimulated monocytes (IC50 56 nM) and blocked IgG- and IgM-mediated antibody production in enriched B cells when stimulated in T-cell dependent (anti-CD40+IL-21) and T-cell independent (TLR-9/CpG and TNP-LPS) pathways. The impact of rilzabrutinib on innate cell pathways was further confirmed by significant dose-dependent inhibition of macrophage and neutrophil-driven passive rat Arthus reaction (P < 0.01 vs vehicle) and antibody-induced murine ITP (P < 0.05 vs vehicle). In a 12-week phase 2 pemphigus vulgaris trial, 54% of patients achieved the primary endpoint, control of disease activity (CDA) on low-dose corticosteroids by week 4, and 73% achieved it by week 12. In the phase 2 trial of ITP patients (median 6 prior therapies), rilzabrutinib 400 mg bid showed rapid and sustained improvement in platelet counts and only grade 1/2-related adverse events1. In responders, platelet counts increased as early as day 8, potentially due to innate immune mechanisms. Collectively, results in both B and innate immune cells provide an initial basis for evaluating rilzabrutinib in IgG4-RD. The ongoing phase 2a study (NCT04520451) is investigating rilzabrutinib 400 mg bid (+tapered GC) vs GC control (3:1) for 12 weeks in IgG4-RD patients refractory to rituximab. The primary objective is to evaluate the safety and ability of rilzabrutinib to induce GC-free remission at week 12. Coupled with known preclinical/clinical findings, mechanistic analyses in this ongoing IgG4-RD study will profile B and other immune cell effects pre-/post-rilzabrutinib dosing to enhance the clinical understanding of rilzabrutinib in IgG4-RD.Conclusion:Studies of rilzabrutinib that show beneficial effects on both B-cell and innate cell pathways provide support for its therapeutic role in immune-mediated diseases and for targeting the underlying pathophysiological effects of IgG4-RD. Effective and safe therapies that rapidly induce and maintain clinical responses, while minimizing the need for continuous GC treatment, remain an unmet need for patients with IgG4-RD.References:[1]Kuter et al. Res Pract Thromb Haemost. 2020;4(suppl 1): PB1318.Disclosure of Interests:Li Long Employee of: Principia Biopharma, a Sanofi Company, Matthew Baker: None declared, Mollie Carruthers: None declared, Alireza Meysami: None declared, Robert Spiera Consultant of: research funding and personal fees for consulting from Chemocentryx, Formation Biologics, Roche-Genentech, and Sanofi, Grant/research support from: research funding fees from BMS, Boehringer Ingelheim, Corbus, GSK, and Inflarx; personal fees from AbbVie, CSL Behring, GSK, and Janssen, Mamatha Reddy Employee of: Principia Biopharma, a Sanofi Company, Marianne Kavanagh Employee of: Principia Biopharma, a Sanofi Company, Michelle Francesco Employee of: Principia Biopharma, a Sanofi Company, Claire Langrish Employee of: Principia Biopharma, a Sanofi Company, Ann Neale Employee of: Principia Biopharma, a Sanofi Company, Puneet Arora Employee of: Principia Biopharma, a Sanofi Company, John H. Stone Consultant of: research funding and personal fees for consulting from Principia and Sanofi

Are Fc Gamma Receptor Polymorphisms Important in HIV-1 Infection Outcomes and Latent Reservoir Size?

Fc gamma receptors (FcγR) are cell surface glycoproteins which trigger specific effector-cell responses when cross-linked with the Fc portions of immunoglobulin (IgG) antibodies. During HIV-1 infection, the course of disease progression, ART response, and viral reservoir size vary in different individuals. Several factors may account for these differences; however, Fc gamma receptor gene polymorphisms, which influence receptor binding to IgG antibodies, are likely to play a key role. FcγRIIa (CD32) was recently reported as a potential marker for latent HIV reservoir, however, this assertion is still inconclusive. Whether FcγR polymorphisms influence the size of the viral reservoir, remains an important question in HIV cure studies. In addition, potential cure or viral suppression methods such as broadly neutralizing antibody (bNAbs) may depend on FcγRs to control the virus. Here, we discuss the current evidence on the potential role played by FcγR polymorphisms in HIV-1 infection, treatment and vaccine trial outcomes. Importantly, we highlight contrasting findings that may be due to multiple factors and the relatively limited data from African populations. We recommend further studies especially in sub-Saharan Africa to confirm the role of FcγRIIa in the establishment of latent reservoir and to determine their influence in therapies involving bNAbs.

Human Cytomegalovirus antagonizes activation of Fcγ receptors by distinct and synergizing modes of IgG manipulation

Human Cytomegalovirus (HCMV) is endowed with multiple highly sophisticated immune evasion strategies. This includes the evasion from antibody mediated immune control by counteracting host Fc-gamma receptor (FcγR) mediated immune control mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC). We have previously shown that HCMV avoids FcγR activation by concomitant expression of the viral Fc-gamma binding glycoproteins (vFcγRs) gp34 and gp68. We now show that gp34 and gp68 bind IgG simultaneously at topologically different Fcγ sites and achieve efficient antagonization of host FcγR activation by distinct but synergizing mechanisms. While gp34 enhances immune complex internalization, gp68 acts as inhibitor of host FcγR binding to immune complexes. In doing so, gp68 induces Fcγ accessibility to gp34 and simultaneously limits host FcγR recognition. The synergy of gp34 and gp68 is compelled by the interfering influence of excessive non-immune IgG ligands and highlights conformational changes within the IgG globular chains critical for antibody effector function.