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Background SARS-CoV-2 induces endothelial damage and activates the complement system. In severe COVID-19 patients, complement split factor C5a is highly elevated leading to inflammation that contributes to multiorgan failure. The anti-C5a monoclonal antibody, Vilobelimab (Vilo), which preserves the membrane attack complex (MAC), was investigated in an adaptively designed, randomized double-blind, placebo (P)-controlled Phase 3 international multicenter study for survival in critically ill COVID-19 patients (pts). Methods COVID-19 pneumonia pts (N=368; Vilo n=177, P n=191), mechanically ventilated within 48 hrs before treatment, received up to 6, 800 mg infusions of Vilo or P on top of standard of care. The primary and main secondary endpoints were 28-day (d) and 60-d all-cause mortality. Results Pts enrolled in the study were on corticosteroids (97%) and anti-coagulants (98%) as standard of care. A smaller proportion (20%) were either continuing or had taken immunomodulators such as tocilizumab and baricitinib prior to receiving Vilo. The 28-d all-cause mortality was 31.7% with Vilo vs 41.6% with P (Kaplan-Meier estimates; Cox regression site-stratified, HR 0.73; 95% CI:0.50-1.06; P=0.094), representing a 23.8% relative mortality reduction. In predefined primary outcome analysis without site stratification, however, Vilo significantly reduced mortality at 28 (HR 0.67; 95% CI:0.48-0.96; P=0.027) and 60 days (HR 0.67; 95% CI:0.48-0.92; P=0.016). Vilo also significantly reduced 28-d mortality in more severe pts with baseline WHO ordinal scale score of 7 (n=237, HR 0.62; 95% CI:0.40-0.95; P=0.028), severe ARDS/PaO2/FiO2 ≤ 100 mmHg (n=98, HR 0.55; 95% CI:0.30-0.98; P=0.044) and eGFR < 60 mL/min/1.73m2 (n=108, HR 0.55; 95% CI:0.31-0.96; P=0.036). Treatment-emergent AEs were 90.9% Vilo vs 91.0% P. Infections were comparable: Vilo 62.9%, P 59.3%. Infection incidence per 100 Pt days were equal. No meningococcal infections were reported. Serious AEs were 58.9% Vilo, 63.5% P. Conclusion Vilo significantly reduced mortality at 28 and 60 days in critically ill COVID-19 pts with no increase in infections suggesting the importance of targeting C5a while preserving MAC. Vilo targets inflammation which may represent an approach to treat sepsis and ARDS caused by other respiratory viruses. Disclosures Alexander Vlaar, MD, PhD, InflaRx GmbH: Advisor/Consultant Maria Habel, PhD, InflaRx GmbH: Stocks/Bonds Claus Thielert, PhD, InflaRx GmbH: Stocks/Bonds James Dickinson, MSc, InflaRx GmbH: Stocks/Bonds simon Rückinger, PhD, InflaRx GmbH: Advisor/Consultant Robert Zerbib, MSc, InflaRx GmbH: Stocks/Bonds Dorothee Neukirchen, PhD, InflaRx GmbH: Stocks/Bonds Korinna Pilz, MD, MSc, InflaRx GmbH: Ownership Interest|InflaRx GmbH: Stocks/Bonds Renfeng Guo, MD, InflaRx GmbH: Board Member|InflaRx GmbH: CSO|InflaRx GmbH: Ownership Interest|InflaRx GmbH: Stocks/Bonds Diederik van de Beek, MD, PhD, InflaRx GmbH: Advisor/Consultant Niels Riedemann, MD, PhD, InflaRx GmbH: Board Member|InflaRx GmbH: CEO|InflaRx GmbH: Ownership Interest|InflaRx GmbH: Stocks/Bonds.

Contains fulltext : 287661.pdf (Publisher’s version ) (Closed access) BACKGROUND: Vilobelimab, an anti-C5a monoclonal antibody, was shown to be safe in a phase 2 trial of invasively mechanically ventilated patients with COVID-19. Here, we aimed to determine whether vilobelimab in addition to standard of care improves survival outcomes in this patient population. METHODS: This randomised, double-blind, placebo-controlled, multicentre phase 3 trial was performed at 46 hospitals in the Netherlands, Germany, France, Belgium, Russia, Brazil, Peru, Mexico, and South Africa. Participants aged 18 years or older who were receiving invasive mechanical ventilation, but not more than 48 h after intubation at time of first infusion, had a PaO(2)/FiO(2) ratio of 60-200 mm Hg, and a confirmed SARS-CoV-2 infection with any variant in the past 14 days were eligible for this study. Eligible patients were randomly assigned (1:1) to receive standard of care and vilobelimab at a dose of 800 mg intravenously for a maximum of six doses (days 1, 2, 4, 8, 15, and 22) or standard of care and a matching placebo using permuted block randomisation. Treatment was not continued after hospital discharge. Participants, caregivers, and assessors were masked to group assignment. The primary outcome was defined as all-cause mortality at 28 days in the full analysis set (defined as all randomly assigned participants regardless of whether a patient started treatment, excluding patients randomly assigned in error) and measured using Kaplan-Meier analysis. Safety analyses included all patients who had received at least one infusion of either vilobelimab or placebo. This study is registered with ClinicalTrials.gov, NCT04333420. FINDINGS: From Oct 1, 2020, to Oct 4, 2021, we included 368 patients in the ITT analysis (full analysis set; 177 in the vilobelimab group and 191 in the placebo group). One patient in the vilobelimab group was excluded from the primary analysis due to random assignment in error without treatment. At least one dose of study treatment was given to 364 (99%) patients (safety analysis set). 54 patients (31%) of 177 in the vilobelimab group and 77 patients (40%) of 191 in the placebo group died in the first 28 days. The all-cause mortality rate at 28 days was 32% (95% CI 25-39) in the vilobelimab group and 42% (35-49) in the placebo group (hazard ratio 0·73, 95% CI 0·50-1·06; p=0·094). In the predefined analysis without site-stratification, vilobelimab significantly reduced all-cause mortality at 28 days (HR 0·67, 95% CI 0·48-0·96; p=0·027). The most common TEAEs were acute kidney injury (35 [20%] of 175 in the vilobelimab group vs 40 [21%] of 189 in the placebo), pneumonia (38 [22%] vs 26 [14%]), and septic shock (24 [14%] vs 31 [16%]). Serious treatment-emergent adverse events were reported in 103 (59%) of 175 patients in the vilobelimab group versus 120 (63%) of 189 in the placebo group. INTERPRETATION: In addition to standard of care, vilobelimab improves survival of invasive mechanically ventilated patients with COVID-19 and leads to a significant decrease in mortality. Vilobelimab could be considered as an additional therapy for patients in this setting and further research is needed on the role of vilobelimab and C5a in other acute respiratory distress syndrome-causing viral infections. FUNDING: InflaRx and the German Federal Government.

Axon formation is a hallmark of initial neuronal polarization. This process is thought to be regulated by enhanced microtubule stability in the subsequent axon and changes in actin dynamics in the future axonal growth cone. Here, we show that the microtubule end-binding proteins cytoplasmic linker protein (CLIP)-115 and CLIP-170 were enriched in the axonal growth cone and extended into the actin-rich domain of the growth cone. CLIPs were necessary for axon formation and sufficient to induce an axon. The regulation of axonal microtubule stabilization by CLIPs enabled the protrusion of microtubules into the leading edge of the axonal growth cone. Moreover, CLIPs positively regulated growth cone dynamics and restrained actin arc formation, which was necessary for axon growth. In fact, in neurons without CLIP activity, axon formation was restored by actin destabilization or myosin II inhibition. Together, our data suggest that CLIPs enable neuronal polarization by controlling the stabilization of microtubules and growth cone dynamics.

Neuronal migration and axon growth, key events during neuronal development, require distinct changes in the cytoskeleton. Although many molecular regulators of polarity have been identified and characterized, relatively little is known about their physiological role in this process. To study the physiological function of Rac1 in neuronal development, we have generated a conditional knock-out mouse, in whichRac1is ablated in the whole brain.Rac1-deficient cerebellar granule neurons, which do not express other Rac isoforms, showed impaired neuronal migration and axon formation bothin vivoandin vitro. In addition,Rac1ablation disrupts lamellipodia formation in growth cones. The analysis of Rac1 effectors revealed the absence of the Wiskott–Aldrich syndrome protein (WASP) family verprolin-homologous protein (WAVE) complex from the plasma membrane of knock-out growth cones. Loss of WAVE function inhibited axon growth, whereas overexpression of a membrane-tethered WAVE mutant partially rescued axon growth inRac1-knock-out neurons. In addition, pharmacological inhibition of the WAVE complex effector Arp2/3 also reduced axon growth. We propose that Rac1 recruits the WAVE complex to the plasma membrane to enable actin remodeling necessary for axon growth.

Axon formation is the initial step in establishing neuronal polarity. We examine here the role of microtubule dynamics in neuronal polarization using hippocampal neurons in culture. We see increased microtubule stability along the shaft in a single neurite before axon formation and in the axon of morphologically polarized cells. Loss of polarity or formation of multiple axons after manipulation of neuronal polarity regulators, synapses of amphids defective (SAD) kinases, and glycogen synthase kinase-3β correlates with characteristic changes in microtubule turnover. Consistently, changing the microtubule dynamics is sufficient to alter neuronal polarization. Application of low doses of the microtubule-destabilizing drug nocodazole selectively reduces the formation of future dendrites. Conversely, low doses of the microtubule-stabilizing drug taxol shift polymerizing microtubules from neurite shafts to process tips and lead to the formation of multiple axons. Finally, local stabilization of microtubules using a photoactivatable analogue of taxol induces axon formation from the activated area. Thus, local microtubule stabilization in one neurite is a physiological signal specifying neuronal polarization.

The establishment of polarity is an essential process in early neuronal development. Although a number of molecules controlling neuronal polarity have been identified, genetic evidence about their physiological roles in this process is mostly lacking. We analyzed the consequences of loss of Cdc42, a central regulator of polarity in multiple systems, on the polarization of mammalian neurons. Genetic ablation of Cdc42 in the brain led to multiple abnormalities, including striking defects in the formation of axonal tracts. Neurons from the Cdc42 null animals sprouted neurites but had a strongly suppressed ability to form axons bothin vivoand in culture. This was accompanied by disrupted cytoskeletal organization, enlargement of the growth cones, and inhibition of filopodial dynamics. Axon formation in the knock-out neurons was rescued by manipulation of the actin cytoskeleton, indicating that the effects of Cdc42 ablation are exerted through modulation of actin dynamics. In addition, the knock-outs showed a specific increase in the phosphorylation (inactivation) of the Cdc42 effector cofilin. Furthermore, the active, nonphosphorylated form of cofilin was enriched in the axonal growth cones of wild-type, but not of mutant, neurons. Importantly, cofilin knockdown resulted in polarity defects quantitatively analogous to the ones seen after Cdc42 ablation. We conclude that Cdc42 is a key regulator of axon specification, and that cofilin is a physiological downstream effector of Cdc42 in this process.

Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.

SummaryNeurites are the characteristic structural element of neurons that will initiate brain connectivity and elaborate information. Early in development, neurons are spherical cells but this symmetry is broken through the initial formation of neurites. This fundamental step is thought to rely on actin and microtubule dynamics. However, it is unclear which aspects of the complex actin behavior control neuritogenesis and which molecular mechanisms are involved. Here, we demonstrate that augmented actin retrograde flow and protrusion dynamics facilitate neurite formation. Our data indicate that a single family of actin regulatory proteins, ADF/Cofilin, provides the required control of actin retrograde flow and dynamics to form neurites. In particular, the F-actin severing activity of ADF/Cofilin organizes space for the protrusion and bundling of microtubules, the backbone of neurites. Our data reveal how ADF/Cofilin organizes the cytoskeleton to drive actin retrograde flow and thus break the spherical shape of neurons.