Efficacy of an experimental gonococcal lipooligosaccharide mimitope vaccine requires terminal complement

A safe and effective vaccine against multidrug-resistant gonorrhea is urgently needed. An experimental peptide vaccine called TMCP2 that mimics an oligosaccharide epitope in gonococcal lipooligosaccharide, when adjuvanted with glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE), elicits bactericidal IgG and hastens clearance of gonococci in the mouse vaginal colonization model. Here, we show that efficacy of TMCP2 requires an intact terminal complement pathway, evidenced by loss of activity in C9 -/- mice or when C7 function was blocked. In conclusion, TMCP2 vaccine efficacy in the mouse vagina requires membrane attack complex. Serum bactericidal activity may serve as a correlate of protection for TMCP2.

Increase in the Complement Activation Product C4d and the Terminal Complement Complex sC5b-9 Is Associated with Disease Severity and a Fatal Outcome in Necrotizing Soft-Tissue Infection

The hyperinflammatory burden is immense in necrotizing soft-tissue infection (NSTI). The complement system is a key during the innate immune response and may be a promising target to reduce the inflammatory response, potentially improving the clinical outcome. However, complement activation and its association to disease severity and survival remain unknown in NSTI. Therefore, we prospectively enrolled patients with NSTI and sampled blood at admission and once daily for the following 3 days. Plasma C4c, C4d, C3bc, and C3dg and the terminal complement complex (TCC) were evaluated using ELISA techniques. In total, 242 patients were included with a median age of 62 years, with a 60% male predominance. All-cause 30-day mortality was 17% (95% confidence interval [CI] 13–23) with a follow-up of &#x3e;98%. C4c and C3dg were negatively correlated with Simplified Acute Physiology Score II (<i>Rho</i> −0.22, <i>p</i> &#x3c; 0.001 and <i>Rho</i> −0.17, <i>p</i> = 0.01). Patients with septic shock (<i>n</i> = 114, 47%) had higher levels of baseline TCC than those in non-shock patients (18 vs. 14, <i>p</i> &#x3c; 0.001). TCC correlated with the Sequential Organ Failure Assessment (SOFA) score (<i>Rho</i> 0.19, <i>p</i> = 0.004). In multivariate Cox regression analysis (adjusted for age, sex, comorbidity, and SOFA score), high baseline C4d (&#x3e;20 ng/mL) and the combination of high C4d and TCC (&#x3e;31 arbitrary units/mL) were associated with increased 30-day mortality (hazard ratio [HR] 3.26, 95% CI 1.56–6.81 and HR 5.12, 95% CI 2.15–12.23, respectively). High levels of both C4d and TCC demonstrated a negative predictive value of 0.87. In conclusion, we found that in patients with NSTI, complement activation correlated with the severity of the disease. High baseline C4d and combination of high C4d and TCC are associated with increased 30-day mortality. Low baseline C4d or TCC indicates a higher probability of survival.

Soliris to Stop Immune-Mediated Death in COVID-19 (SOLID-C19)—A Compassionate-Use Study of Terminal Complement Blockade in Critically Ill Patients with COVID-19-Related Adult Respiratory Distress Syndrome

Eculizumab, a terminal complement (C5)-inhibiting monoclonal antibody, was administered in five mechanically ventilated patients in life-threatening condition due to COVID-19-related acute respiratory distress syndrome (ARDS) between 23 March 2020 and 3 April 2020. Their clinical progress was monitored. The primary endpoint was mortality. One patient was excluded while two passed away. The remaining two patients survived. At the time of this study, the mortality rate in mechanically ventilated COVID-19 patients suffering from ARDS receiving the standard of care as their therapeutic regimen was reportedly as high as 97%. This pilot study demonstrates a 50% mortality rate in patients receiving eculizumab therapy.

A Challenging Case of Atypical Hemolytic Uremic Syndrome in a Young African American Patient

Background It is known that malignant hypertension (mHTN) and thrombotic microangiopathy (TMA) commonly coexist. Deciding which phenomenon preceded the other remains a clinical dilemma, specifically in African American patients. However, making that determination is of utmost importance because the management will be different, and that can have dramatic effects on prognosis and outcomes. Herein, we report a case of atypical hemolytic uremic syndrome (aHUS) presenting as mHTN. Case Presentation A 35-year-old African American male with known history of hypertension, presented with nausea, vomiting, and diarrhea for four days. He also reported fatigue and exertional shortness of breath. Upon presentation, his blood pressure was 260/160 mmHg, otherwise physical exam was unremarkable. Initial work up showed hemoglobin of 8.8 g/dL (baseline 13.5), platelet count of 21,000/mL (baseline 250,000), serum creatinine of 16.99 mg/dL (baseline 0.99), MCV (84 fl), increased reticulocyte production index (3.58), increased LDH (1709 U/L), undetectable haptoglobin, and numerous schistocytes on peripheral blood smear. He was admitted as a case of hypertensive emergency and TMA. IV labetalol and hemodialysis were started. Given his gastrointestinal symptoms; stool for Shigella and E.Coli O157:H7 were checked and they were negative. Given the severity of his hematologic derangements and difficult to control blood pressure, we decided to proceed with renal biopsy to rule out primary aHUS which showed thrombotic microangiopathy, global glomerulosclerosis, moderate interstitial fibrosis and tubular atrophy suggestive of aHUS or rheumatologic disorders like systemic sclerosis and arguing against malignant HTN as the sole player. ANA and anti-Scl-70 antibodies were negative. Final impression was aHUS by exclusion, and patient received meningococcal vaccines (Menactra and Bexsero) in preparation to start eculizumab. aHUS genetic panel was sent which came back equivocal as it showed mutations of unknown significance (homozygous missense mutation in the MASP2 gene and 2 heterozygous mutations in the C2 gene). He was started on eculizumab 900 mg weekly for 4 weeks then 1200 mg biweekly starting week 5. He was seen in the office 2 months after initial presentation and receiving 5 doses of Eculizumab. His kidney function showed improvement with &gt; 2 liters of urine output daily, blood pressure was better controlled. A decision by nephrology was made to give him a break from dialysis and remains dialysis-free a year later. Discussion aHUS is a rare disorder with an estimated prevalence of seven per one million children in Europe. It causes uninhibited activation of complement factors that leads to renal endothelial damage and activation of coagulation cascade leading to TMA. The diagnosis of aHUS requires the fulfillment of the classical triad (microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure) with a positive gene mutation or antibodies to complement factors. However, absence of these mutations or antibodies, as in the presented case, do not exclude the diagnosis. The early diagnosis of aHUS is necessary for treatment with eculizumab, a monoclonal antibody against C5 to block the terminal complement cascade. Kidney biopsy can be helpful in equivocal cases especially if it shows only the typical changes of malignant hypertension which essentially rules out aHUS. Hypertension with concurrent TMA is treated with strict BP control which is often enough to resolve TMA features and restores renal function, at least partially. On the contrary, aHUS causing severe HTN needs more sophisticated testing and blockade of the terminal complement component to improve outcome; that's why the distinction of which one is the primary process is of utmost importance. Our case emphasizes the importance of having low threshold for testing for aHUS in patients with mHTN and TMA, especially in African American patients where malignant HTN is known to happen more commonly, and to notice the subtle hints that may help in this distinction, such as profound hemolysis or thrombocytopenia out of proportion to what one would expect from mHTN alone. Early recognition of aHUS may save a patient's kidney. Disclosures No relevant conflicts of interest to declare.

A Randomized, Double-Blind, Single-Dose Phase 1 Comparative Pharmacokinetic Study Comparing SB12 (Eculizumab Biosimilar) with Reference Eculizumab in Healthy Volunteers

Background: SB12 has been developed as a biosimilar of the reference product (RP) eculizumab. Eculizumab is a humanized monoclonal antibody (IgG2/4 kappa immunoglobulin) that binds to the human C5 complement protein with high affinity. Binding to this protein blocks its cleavage into C5a and C5b, thereby inhibiting terminal complement-mediated intravascular haemolysis. It is currently indicated for the treatment of patients with paroxysmal nocturnal haemoglobinuria, atypical haemolytic uremic syndrome (aHUS), refractory generalized Myasthenia Gravis (gMG) and Neuromyelitis Optica Spectrum Disorder (NMOSD). Objectives: To demonstrate pharmacokinetic (PK) equivalence and evaluate pharmacodynamic (PD), safety, tolerability, and immunogenicity profiles between SB12 and the RP eculizumab. Methods: This was a double-blind, three-arm, parallel group, and single-dose study in healthy subjects, between 18-55 years of age, randomized in a ratio of 1:1:1 to receive a single 300 mg dose of either SB12, European Union (EU) sourced eculizumab, or United States of America (US) sourced eculizumab via intravenous (IV) infusion for 35 minutes. Blood samples for PK and PD analysis were collected over 64 days. The primary objective of this study was to demonstrate PK similarity between the investigational products (IPs), as assessed by area under the concentration-time curve from time zero to infinity (AUC inf). Secondary PK endpoints included area under the concentration-time curve from time zero to the last quantifiable concentration (AUC last) and maximum observed concentration (C max). Equivalence for the primary endpoint (AUC inf) was determined if 90% confidence intervals (CIs) for the ratio of geometric least squared means (LSMeans) of SB12 to EU sourced eculizumab, SB12 to US sourced eculizumab, and EU sourced eculizumab to US sourced eculizumab was within the equivalence margin of 80.00% to 125.00%, respectively. Other objectives for the study were to evaluate safety, tolerability, immunogenicity, and PD profiles for the IPs. Results: A total of 240 subjects (80 in each treatment group) were enrolled. Back transformation provided the geometric LSMean ratio for the comparison of SB12/EU sourced eculizumab, SB12/US sourced eculizumab and EU sourced eculizumab/US sourced eculizumab for AUC inf were 99.1 % (95.41,102.85), 95.1 % (91.40, 99.04), and 96.0 % (92.16, 100.10), respectively. The corresponding 90% CI was within the pre-defined equivalence margin of 80.00-125.00%, indicating that the each of two treatments are bioequivalent. The profiles of mean terminal complement activity and mean change from baseline of complement activity were superimposable following administration of SB12, EU sourced eculizumab, and US sourced eculizumab. There was a rapid decrease in the complement activity at the end of infusion and then a slow restoration. There was no non-responder in the aspect of the measured complement activity after treatment. There were no deaths or discontinuation of IP due to treatment-emergent adverse events (TEAEs) during the study. Two serious adverse events (SAEs) (renal colic in the SB12 treatment group and back pain in the US eculizumab treatment group) were reported, in 2 subjects. Both events were considered not related to the IP. The proportion of subjects who experienced TEAEs were similar between the SB12, EU sourced eculizumab, and US sourced eculizumab treatment groups (70.0%, 65.0%, and 71.3% of subjects, respectively). The overall incidence of subjects with post-dose anti-drug antibodies (ADA) to eculizumab was 2 (2.5%), 1 (1.3%), and 0 (0.0%) subjects in the SB12, EU sourced eculizumab, and US sourced eculizumab treatment groups, respectively. There was no significant difference between treatment groups. None of the subjects with post-dose ADA to eculizumab had a positive result for neutralizing antibodies. Conclusion: The Phase I study demonstrated PK bioequivalence and showed comparable PD, safety, immunogenicity between SB12 and the RP eculizumab. Disclosures Lee: Samsung Bioepis, Co., Ltd.: Current Employment. Jang: Samsung Bioepis, Co., Ltd.: Current Employment. Kim: Samsung Bioepis, Co., Ltd.: Current Employment. Jeong: Samsung Bioepis, Co., Ltd.: Current Employment. Lee: Samsung Bioepis, Co., Ltd.: Current Employment. Jung: Samsung Bioepis, Co., Ltd.: Current Employment. Demichelis: Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol/Celgene: Consultancy, Speakers Bureau; AMGEN: Consultancy, Speakers Bureau; ASH: Research Funding; Jazz: Consultancy; Gilead: Consultancy; Astellas: Consultancy; Abbvie: Consultancy, Speakers Bureau.

Eculizumab Pharmacokinetics and Pharmacodynamics in Patients With Neuromyelitis Optica Spectrum Disorder

Objective: To investigate the pharmacokinetics and pharmacodynamics of the approved 900/1,200 mg dosing regimen for the terminal complement component 5 (C5) inhibitor eculizumab in patients with neuromyelitis optica spectrum disorder (NMOSD).Methods: Data were analyzed from 95 patients with aquaporin-4-IgG-positive NMOSD who received eculizumab during the PREVENT study (ClinicalTrials.gov: NCT01892345). Relationships were explored between eculizumab exposure and free complement C5 concentrations, terminal complement activity, and clinical outcomes.Results: Pharmacokinetic data were well-described by a two-compartment model with first-order elimination, and time-variant body-weight and plasmapheresis/plasma exchange effects. Steady-state serum eculizumab concentrations were achieved by Week 4 and were sustained, with serum trough eculizumab concentrations maintained above the 116 μg/ml threshold for complete complement inhibition throughout 168 weeks of treatment in all post-baseline samples from 89% of patients. Complete inhibition of terminal complement was achieved at Day 1 peak and pre-dosing trough eculizumab concentration in nearly all post-baseline samples assessed (free C5 &lt;0.5 μg/ml in all post-baseline samples from 96% of patients; in vitro hemolysis &lt;20% in all post-baseline samples from 93% of patients). Kaplan–Meier survival analysis of time to first relapse showed separation of eculizumab-treated patients from those receiving placebo, but no separation based on eculizumab exposure quartile, indicating an optimized dose regimen with maximized efficacy.Conclusions: The approved eculizumab dosing regimen (900/1,200 mg) for adults with aquaporin-4-IgG-positive NMOSD is confirmed by rigorous quantitative model-based analysis of exposure–response. The data demonstrate that eculizumab's mechanism of action translates into clinical effect by achieving rapid, complete, and sustained terminal complement inhibition.

Novel Selection Approaches to Identify Antibodies Targeting Neoepitopes on the C5b6 Intermediate Complex to Inhibit Membrane Attack Complex Formation

The terminal pathway of complement is implicated in the pathology of multiple diseases and its inhibition is, therefore, an attractive therapeutic proposition. The practicalities of inhibiting this pathway, however, are challenging, as highlighted by the very few molecules in the clinic. The proteins are highly abundant, and assembly is mediated by high-affinity protein–protein interactions. One strategy is to target neoepitopes that are present transiently and only exist on active or intermediate complexes but not on the abundant native proteins. Here, we describe an antibody discovery campaign that generated neoepitope-specific mAbs against the C5b6 complex, a stable intermediate complex in terminal complement complex assembly. We used a highly diverse yeast-based antibody library of fully human IgGs to screen against soluble C5b6 antigen and successfully identified C5b6 neoepitope-specific antibodies. These antibodies were diverse, showed good binding to C5b6, and inhibited membrane attack complex (MAC) formation in a solution-based assay. However, when tested in a more physiologically relevant membrane-based assay these antibodies failed to inhibit MAC formation. Our data highlight the feasibility of identifying neoepitope binding mAbs, but also the technical challenges associated with the identification of functionally relevant, neoepitope-specific inhibitors of the terminal pathway.

The protease SplB of Staphylococcus aureus targets host complement components and inhibits complement-mediated bacterial opsonophagocytosis

Staphylococcus aureus is an opportunistic pathogen that can cause life-threatening infections, particularly in immunocompromised individuals. The high-level virulence of S. aureus largely relies on its diverse and variable collection of virulence factors and immune-evasion proteins, including the six serine protease-like proteins SplA-SplF. Spl proteins are expressed by most clinical isolates of S. aureus , but little is known about the molecular mechanisms by which these proteins modify the host’s immune response for the benefit of the bacteria. Here, we identify SplB as a protease that inactivates central human complement proteins, i.e., C3, C4, and the activation fragments C3b and C4b, by preferentially cleaving their α-chains. SplB maintained its proteolytic activity in human serum, degrading C3 and C4. SplB further cleaved the components of the terminal complement pathway, C5, C6, C7, C8, and C9. By contrast, the important soluble human complement regulators, Factor H and C4BP, as well as C1q, were left intact. Thereby SplB reduced C3b-mediated opsonophagocytosis by human neutrophils as well as C5b-9 deposition on the bacterial surface. In conclusion, we identified the first physiological substrates of the S. aureus extracellular protease SplB. This enzyme inhibits all three complement pathways and blocks opsonophagocytosis. Thus, SplB can be considered as a novel staphylococcal complement-evasion protein. Importance Success of bacterial pathogens in immunocompetent humans depends on control and inactivation of host immunity. S aureus , like many other pathogens, efficiently blocks host complement attack early in infection. Aiming to understand the role of the S. aureus -encoded orphan proteases SplA-SplD, we asked whether these proteins play a role in immune escape. We found that SplB inhibits all three-complement activation pathways as well as the lytic terminal complement pathway. This blocks opsonophagocytosis of the bacteria by neutrophils. We also clarified the molecular mechanisms: SplB cleaves the human complement proteins C3, C4, C5, C6, C7, C8 C9 as well as Factor B, but not the complement inhibitors Factor H and C4BP. Thus we identified the first physiological substrates of the extracellular protease SplB of S. aureus and characterize SplB as a novel staphylococcal complement-evasion protein.