In vitro, classical complement activation differs by disease severity and between SARS-CoV-2 antigens

Antibodies specific for the spike glycoprotein (S) and nucleocapsid (N) SARS-CoV-2 proteins are typically present during severe COVID-19, and induced to S after vaccination. The binding of viral antigens by antibody can initiate the classical complement pathway. Since complement could play pathological or protective roles at distinct times during SARS-CoV-2 infection we determined levels of antibody-dependent complement activation along the complement cascade. Here, we used an ELISA assay to assess complement protein binding (C1q) and the deposition of C4b, C3b, and C5b to S and N antigens in the presence of anti-SARS-CoV-2 antibodies from different test groups: non-infected, single and double vaccinees, non-hospitalised convalescent (NHC) COVID-19 patients and convalescent hospitalised (ITU-CONV) COVID-19 patients. C1q binding correlates strongly with antibody responses, especially IgG1 levels. However, detection of downstream complement components, C4b, C3b and C5b shows some variability associated with the antigen and subjects studied. In the ITU-CONV, detection of C3b-C5b to S was observed consistently, but this was not the case in the NHC group. This is in contrast to responses to N, where median levels of complement deposition did not differ between the NHC and ITU-CONV groups. Moreover, for S but not N, downstream complement components were only detected in sera with higher IgG1 levels. Therefore, the classical pathway is activated by antibodies to multiple SARS-CoV-2 antigens, but the downstream effects of this activation may differ depending on the specific antigen targeted and the disease status of the subject.

Activation of Intracellular Complement in Lungs of Patients With Severe COVID-19 Disease Decreases T-Cell Activity in the Lungs

A novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), arose late in 2019, with disease pathology ranging from asymptomatic to severe respiratory distress with multi-organ failure requiring mechanical ventilator support. It has been found that SARS-CoV-2 infection drives intracellular complement activation in lung cells that tracks with disease severity. However, the cellular and molecular mechanisms responsible remain unclear. To shed light on the potential mechanisms, we examined publicly available RNA-Sequencing data using CIBERSORTx and conducted a Ingenuity Pathway Analysis to address this knowledge gap. In complement to these findings, we used bioinformatics tools to analyze publicly available RNA sequencing data and found that upregulation of complement may be leading to a downregulation of T-cell activity in lungs of severe COVID-19 patients. Thus, targeting treatments aimed at the modulation of classical complement and T-cell activity may help alleviate the proinflammatory effects of COVID-19, reduce lung pathology, and increase the survival of COVID-19 patients.

Evidence of Classical Complement Pathway Involvement in a Subset of Patients with Warm Autoimmune Hemolytic Anemia

Introduction: Autoimmune Hemolytic Anemia (AIHA) is caused by autoantibodies that react with red blood cells (RBCs) resulting in predominantly extravascular hemolysis in an FcR and/or complement-dependent manner. In warm AIHA (wAIHA), autoantibodies are generally of the IgG isotype, while in cold agglutinin disease (CAD) they are predominantly of the IgM isotype. It is well established that the classical complement cascade is critical for the pathogenesis of CAD based on therapeutic clinical studies. Published data also suggest that complement activation plays a role in wAIHA, although it is not clear which patients would most benefit from complement-based therapy. To help address this question, we utilized an assay that measures the ability of autoantibodies in patient sera to induce complement deposition on the surface of donor RBCs (based on Meulenbroek, et al., 2015). Methods: Sera were collected retrospectively from 12 wAIHA patients whose direct antiglobulin tests (DAT) were either IgG+/C3+ or IgG+/C3-. Sera retrospectively collected from two CAD patients were used as positive controls. Individual patient sera were examined in the in vitro complement deposition assay using RBCs from type O+ healthy donors. RBCs and sera were incubated at 37 oC in the presence of either EDTA or an inhibitory antibody against C1q as inhibitors of the classical pathway. RBCs were then stained and processed by flow cytometry to determine the level of C4 deposition. Results: Sera from both CAD patients deposited C4 on the surface of ~70% of healthy human RBCs in vitro. Four out of twelve (33%) sera from wAIHA patients displayed this activity, and all four of these patients were identified as IgG+/C3+ on DAT. Complement deposition ranged from ~10-60% of the RBCs in wAIHA, suggesting heterogeneity in antibody activity for complement deposition in sera from wAIHA patients. Addition of EDTA or an inhibitory antibody against C1q fully blocked deposition of C4 on RBCs by wAIHA sera, indicating dependence of the classical complement pathway. These results indicate differences in the frequency of classical pathway involvement in CAD versus wAIHA and may help identify a subset of wAIHA patients most likely to respond to anti-C1q therapy. Conclusions: The hypothesis of classical complement cascade involvement in wAIHA disease in a subset of patients is supported by our results. Critically, complement deposition on the surface of cells by anti-C1q prevented the deposition of a downstream complement marker, C4. Inhibition of C1q has been shown to block activation of all downstream classical complement components, including C3b and C4b involved in extravascular hemolysis and C5b involved in direct cell lysis. The therapeutic potential of blocking classical complement pathway activity in wAIHA is currently being evaluated in an ongoing Phase 2 interventional trial (NCT04691570) assessing efficacy of an anti-C1q drug candidate in wAIHA patients, focusing on those with evidence of classical complement pathway activity. Disclosures Teigler: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Low: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Rose: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Cahir-Mcfarland: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Yednock: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Kroon: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Keswani: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Barcellini: Novartis: Honoraria; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria; Incyte: Membership on an entity's Board of Directors or advisory committees.

Safety, Tolerability, and Clinical Pharmacology of ANX009, an Inhibitory Antibody Fab Fragment Against C1q, Administered Subcutaneously to Healthy Volunteers

Certain autoantibodies that bind to tissue antigens or that deposit in tissues as a component of immune complexes can activate the classical complement cascade, leading to inflammation and tissue damage. As the initiating molecule of the classical complement cascade, C1q is an attractive target for preventing complement activation and its multiple tissue-damaging effects. ANX009 is an antigen binding fragment (Fab) of a humanized antibody against C1q that inhibits C1q substrate interactions and fully blocks activation of all downstream classical complement components. While inhibiting the classical cascade, ANX009 leaves the lectin and alternative complement pathways intact for their normal immune functions. ANX009 is formulated for subcutaneous (SC) administration and is designed for treatment of blood-based and vascular antibody-mediated autoimmune diseases, such as autoimmune hemolytic anemia (AIHA) and lupus nephritis, where complement activation is a key component of disease pathology. A phase 1 first-in-human single ascending dose (SAD) and multiple ascending dose (MAD) study of ANX009 with subcutaneous administration was conducted in 48 healthy volunteers (NCT04535752). Four SAD cohorts were enrolled followed by two MAD cohorts evaluating daily dosing for 7 days or twice weekly dosing x 4 doses. Each cohort had eight participants randomized in a 6:2 active:placebo ratio. Safety and tolerability were assessed, along with serum pharmacokinetics (unbound drug), pharmacodynamics (unbound C1q target), and an ex vivo measure of C1q activity (CH 50 hemolysis of antibody-sensitized sheep red blood cells). All dose levels were well-tolerated. No drug-related safety signals, dose-limiting toxicities, serious adverse events, or adverse events leading to discontinuations were observed. Mild, transient, local injection site reactions were observed. A clear dose-response relationship was observed in SAD cohorts. Negligible reduction in free C1q was observed in the two lowest dose cohorts. A maximum mean reduction in free C1q of 80% was observed at 48 hours post-dose at the third dose level, and full reduction of free C1q through 72 hours was observed at the highest dose level. Similarly, full reduction of free C1q was observed in the MAD cohort with daily dosing as well as in the second MAD cohort with twice weekly dosing. Full reduction of C1q was maintained for 4 days following the last dose in the second MAD cohort. Ex vivo functional activity of C1q was completely inhibited in close correspondence with free C1q levels. Combined safety, tolerability, and clinical pharmacology results from this phase 1 study support advancement of ANX009 to studies in patients with complement-mediated autoimmune disorders. Disclosures Grover: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Teigler: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Radomile: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Rose: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Yednock: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Keswani: Annexon Inc: Current Employment, Current equity holder in publicly-traded company.

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.

1000. Serotype 3 pneumococci evade activation of the classical complement pathway

Background Complement classical pathway (CCP) activation is the major mechanism leading to opsonophagocytic pneumococcal killing. Following immunization with 13-valent pneumococcal conjugate vaccine (PCV13), opsonophagocytic titers are lowest against serotype 3 among the 13 vaccine serotypes. Post licensure surveillance indicated early declines in serotype 3 invasive pneumococcal disease (IPD) were not sustained over time Methods Using flow cytometry, we measured C3 and C4 deposition on serotype 3 strains from children with IPD or nasopharyngeal [NP] carriage, and analyzed by clade. C4 deposition is an indicator of CCP, while C3 deposition is common to all complement pathways. We measured C3/C4 deposition on serotype 3 pneumococcal strains incubated with antibody depleted complement alone or with complement and the following antibodies: mouse monoclonal anti-capsular IgG or IgM, rabbit polyclonal serotype 3 antisera (IgG + IgM) [RPS3A] and RPS3A combined with anti-rabbit IgM, which blocks IgM function, leaving only polyclonal IgG Results Serotype 3 strains demonstrated high variability in C3 binding when incubated with complement alone. RPS3A (containing both IgM+IgG) and monoclonal IgM activated CCP in all strains. Anti- serotype 3 monoclonal IgG and polyclonal IgG demonstrated absent or limited CCP activation; but activated alternative pathway in some strains. When analyzing complement deposition by clade, a lower proportion of clade II NP serotype 3 strains bound C3 when incubated with complement or monoclonal IgG, compared to clade Ia NP strains. Differences between clade Ia and II IPD strains were not apparent. Conclusion Serotype 3 strains did not demonstrate activation of the CCP in the presence IgG and varied in C3 deposition. Pneumococcal strains that evade CCP activation may be less sensitive to opsonophagocytosis. Our findings suggest a mechanism by which serotype 3 carriage and disease may persist despite immunization with conjugate vaccine containing serotype 3 polysaccharide. Disclosures Rotem Lapidot, MD MSCI, Pfizer (Consultant, Grant/Research Support, Advisor or Review Panel member) Mario Ramirez, PhD, GlaxoSmithKline (Advisor or Review Panel member)Merck Sharp & Dohme (Advisor or Review Panel member)Pfizer (Speaker’s Bureau) Ingrid L. Scully, PhD, Pfizer (Employee, Shareholder) Bradford D. Gessner, MD, MPH, Pfizer Inc. (Employee) stephen pelton, MD, Merck Vaccines (Advisor or Review Panel member, Research Grant or Support)Pfizer, Inc. (Consultant, Advisor or Review Panel member, Research Grant or Support)Sanofi pasteur (Advisor or Review Panel member, Research Grant or Support, DSMB)Seqirus (Consultant)

Microglial TREM2 Mitigates Inflammatory Responses and Neuronal Apoptosis in Angiotensin II-Induced Hypertension in Middle-Aged Mice

Growing evidence suggests that hypertension and aging are prominent risk factors for the development of late-onset Alzheimer’s disease (LOAD) by inducement of neuroinflammation. Recent study showed that neuroinflammation via activated microglia induces reactive astrocytes, termed A1 astrocytes, that highly upregulate numerous classical complement cascade genes that are destructive to neurons in neurodegeneration diseases. Moreover, triggering receptor expressed on myeloid cells 2 (TREM2) is considered as one of the strongest single-allele genetic risk factors and plays important roles in neuroinflammation for LOAD. However, the mechanisms of microglia in the regulation of A1 astrocytic activation are still not clear. We introduced angiotensin II-induced hypertension in middle-aged mice and found that hypertension-upregulated TREM2 expression and A1 astrocytic activation were involved in neuroinflammation in the animal models used in this study. The in vitro results revealed that overexpression of microglial TREM2 not only mitigated microglial inflammatory response but also had salutary effects on reverse A1 astrocytic activation and neuronal toxicity.