Zilucoplan in patients with acute hypoxic respiratory failure due to COVID-19 (ZILU-COV) : a structured summary of a study protocol for a randomised controlled trial

Objectives: Zilucoplan (complement C5 inhibitor) has profound effects on inhibiting acute lung injury post COVID-19, and can promote lung repair mechanisms that lead to improvement in lung oxygenation parameters. The purpose of this study is to investigate the efficacy and safety of Zilucoplan in improving oxygenation and short- and long-term outcome of COVID-19 patients with acute hypoxic respiratory failure. Trial design: This is a phase 2 academic, prospective, 2:1 randomized, open-label, multi-center interventional study. Participants: Adult patients (>= 18y old) will be recruited at specialized COVID-19 units and ICUs at 9 Belgian hospitals. The main eligibility criteria are as follows: 1) Inclusion criteria: a. Recent (>= 6 days and <= 16 days) SARS-CoV-2 infection. b. Chest CT scan showing bilateral infiltrates within the last 2 days prior to randomisation. c. Acute hypoxia (defined as PaO2/FiO(2) below 350 mmHg or SpO2 below 93% on minimal 2 L/min supplemental oxygen). d. Signs of cytokine release syndrome characterized by either high serum ferritin, or high D-dimers, or high LDH or deep lymphopenia or a combination of those. 2) Exclusion criteria: e. Mechanical ventilation for more than 24 hours prior to randomisation. f. Active bacterial or fungal infection. g. History of meningococcal disease (due to the known high predisposition to invasive, often recurrent meningococcal infections of individuals deficient in components of the alternative and terminal complement pathways). Intervention and comparator: Patients in the experimental arm will receive daily 32,4 mg Zilucoplan subcutaneously and a daily IV infusion of 2g of the antibiotic ceftriaxone for 14 days (or until hospital discharge, whichever comes first) in addition to standard of care. These patients will receive additional prophylactic antibiotics until 14 days after the last Zilucoplan dose: hospitalized patients will receive a daily IV infusion of 2g of ceftriaxone, discharged patients will switch to daily 500 mg of oral ciprofloxacin.The control group will receive standard of care and a daily IV infusion of 2g of ceftriaxone for 1 week (or until hospital discharge, whichever comes first), to control for the effects of antibiotics on the clinical course of COVID-19. Main outcomes: The primary endpoint is the improvement of oxygenation as measured by mean and/or median change from pre-treatment (day 1) to post-treatment (day 6 and 15 or at discharge, whichever comes first) in PaO2/FiO(2) ratio, P(A-a)O-2 gradient and a/A PO2 ratio.(PAO(2)= Partial alveolar pressure of oxygen, PaO2=partial arterial pressure of oxygen, FiO(2)=Fraction of inspired oxygen). Randomisation: Patients will be randomized in a 2:1 ratio (Zilucoplan: control). Randomization will be done using an Interactive Web Response System (REDCap). Blinding (masking): In this open-label trial neither participants, caregivers, nor those assessing the outcomes will be blinded to group assignment. Numbers to be randomised (sample size): A total of 81 patients will be enrolled: 54 patients will be randomized to the experimental arm and 27 patients to the control arm. Trial Status: ZILU-COV protocol Version 4.0 (June 10 2020). Participant recruitment started on June 23 2020 and is ongoing. Given the uncertainty of the pandemic, it is difficult to predict the anticipated end date. Trial registration: The trial was registered on Clinical Trials.gov on May 11(th), 2020 (ClinicalTrials.gov Identifier: NCT04382755) and on EudraCT (Identifier: 2020-002130-33). Full protocol: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol

Experimental model of autoimmune thrombocytopenia in the mouse : analysis of autoreactive antibodies and of T lymphocyte-dependent pathogenic mechanisms

Self-recognition is an important aspect of homeostasis and is essential for normal physiological processes and for the regulation of the immune system, contributing to controlled production of antibodies and accelerated elimination of aged autoantigens or cells. In some cases, however, an imbalance in this homeostatic state can occur as a result of environmental or genetic triggers. This imbalance may ultimately lead to autoimmunity and clinically significant diseases. For example, systemic lupus erythematosus, autoimmune hepatitis, rheumatoid arthritis, thyroiditis, myasthenia gravis, type I diabetes and many other diseases were shown to have an autoimmune etiology. Two such diseases are autoimmune thrombocytopenia and post-transfusion purpura in which circulating platelets are targeted and are prematurely destroyed by the immune system. Many animal models have been developed and used to investigate the pathophysiological mechanisms involved in the onset and regulation of these diseases. Such models have clearly contributed to narrowing the translational gap between mouse and man although none of these models fully recapitulate the whole extent of the human situation. Thus, a novel murine model was developed in our laboratory which consisted of the immunisation of mice with rat platelets. These immunised mice developed moderate and transient thrombocytopenia which resolved spontaneously despite continuous immunisations. By using this model we could gain some insight into the mechanisms involved in this process. Firstly, we generated monoclonal antiplatelet autoantibodies from these mice and showed that these were of the IgM isotype and displayed pan-reactivity, as they not only reacted to mouse platelets but also to those of rats and humans. We also confirmed that these antibodies, when injected into naïve mice, were responsible for the observed thrombocytopenia by a mechanism likely involving enhanced phagocytosis of the circulating platelets. In addition, these autoantibodies impeded normal platelet function possibly enhancing further their pathogenicity. The way in which both autoantibodies impaired platelet function and their specificity towards a ~95kDa and ~150kDa antigen suggested that they targeted two important platelet receptors, namely glycoprotein IIIa and glycoprotein Ib. Consistently, autoantibodies targeting both these receptors are frequently found in autoimmune thrombocytopenia and post-transfusion purpura. Secondly, we found that the initiation of this autoimmune response was highly dependent on CD4+ T lymphocytes that reacted to rat but not mouse platelet epitopes. Upon stimulation with rat platelets, these primed T lymphocytes proliferated and secreted high levels of IFN-γ and lower levels of IL-4, IL-5, IL-9 and IL-17A, which was indicative of a predominant Th1 response in immunised mice over the Th2 or Th17 responses. Thus, it may be postulated that the activation of anti-rat platelet Th1 lymphocytes initiates the autoimmune response by two distinct mechanisms: (1) by providing adequate help to B lymphocytes that in turn can secrete cross-reactive anti-platelet antibodies that bind not only to the donor rat platelets but also to the autologous mouse platelets and (2) by enhancing macrophage phagocytic capacity of these opsonised platelets by IFN-γ secretion. Finally, CD4+CD25+ expressing T cells were shown to regulate both the induction and the spontaneous remission of the thrombocytopenia in immunised mice. A lack of these regulatory T cells resulted in more severe and prolonged thrombocytopenia which was not correlated with an increased binding of antibodies to circulating platelets. Likewise, administration of regulatory T cells to naïve animals protected these mice from subsequent immunisations. We hypothesised that the regulation of this autoimmune response was exerted at the B cell level but may also, at least in part, be directed towards T cells.La reconnaissance du soi est un aspect important de l’homéostasie, est essentielle pour des processus physiologiques normaux et pour la régulation du système immunitaire. Elle contribue ainsi à la production contrôlée d’anticorps et à l’élimination accélérée d’auto antigènes ou de cellules sénescentes. Néanmoins, dans certaines circonstances, un déséquilibre homéostatique peut se produire suite à un facteur génétique ou environnemental. Ce déséquilibre peut ultérieurement mener à l’auto-immunité et à des maladies cliniquement significatives. Par exemple, le lupus érythémateux disséminé, l’hépatite auto-immune, l’arthrite rhumatoïde, la thyroïdite, la myasthénie, le diabète de type I,… ont tous une étiologie auto-immune. Deux maladies de ce type sont la thrombocytopénie auto-immune et le purpura post-transfusionnel dans lesquels les plaquettes sanguines sont la cible du système immunitaire et sont détruites prématurement dans l’organisme. Un grand nombre de modèles animaux ont été développés pour mettre en évidence les mécanismes pathophysiologiques impliqués dans l’apparition et la régulation de ces maladies. De tels modèles ont clairement contribués à accroître la connaissance et la compréhension de tels mécanismes. Cependant, aucun de ces modèles ne récapitule pleinement la maladie telle qu’observée chez l’homme. C’est la raison pour laquelle un nouveau modèle murin a été développé dans notre laboratoire, consistant en l’immunisation de souris par des plaquettes de rats. Ces souris immunisées développent une thrombopénie modérée et transitoire qui se résorbe spontanément malgré des immunisations constantes. En utilisant ce modèle, il est possible de comprendre les mécanismes pathologiques impliqués dans l’initiation et la régulation de la réponse auto-immune. Tout d’abord, nous avons utilisé ce modèle murin pour en dériver des auto anticorps monoclonaux. L’analyse de ces auto anticorps a démontré qu’ils étaient de type IgM et avaient une spécificité croisée pour les plaquettes humaines, de rats et de souris. Nous avons également confirmé que ces auto anticorps, lorsqu’ils sont injectés dans des souris naïves, sont responsables de la thrombopénie en induisant, très probablement, une phagocytose accrue des plaquettes circulantes. En outre, ces autoanticorps ont la capacité d’entraver la fonction plaquettaire et, ce faisant, peuvent augmenter leur pathogenicité. La façon dont ces auto anticorps inhibent la fonction plaquettaire et leur spécificité envers des antigènes de ~95kDa et ~150kDa suggère qu’ils reconnaissent deux récepteurs plaquettaires importants, à savoir la glycoprotéine IIIa et la glycoprotéine Ib. Il est intéressant de noter que des auto anticorps ciblant ces deux récepteurs sont fréquemment impliqués dans la thrombopénie auto-immune et dans le purpura post-transfusionnel. Ensuite, nous avons démontré que l’initiation de la réponse auto-immune est dépendante de lymphocytes T CD4+ qui ont une spécificité envers des épitopes plaquettaires de rats mais pas de souris. En effet, lorsque ces lymphocytes T sont stimulés avec des plaquettes de rats, ils prolifèrent et secrètent de grandes quantités d’IFN-γ et, dans une moindre mesure, d’IL-4, d’IL-5, d’IL-9 et d’IL-17A. Une telle sécrétion d’IFN-γ est plutôt indicative d’une réponse Th1 prédominante dans des souris immunisées plutôt que d’une réponse Th2 ou Th17. Ainsi, il peut être postulé que l’activation de lymphocytes Th1, qui ont une réactivité pour des plaquettes de rats, induit la réponse auto-immune par deux mécanismes distincts : (1) en apportant une aide adéquate à des lymphocytes B qui peuvent, à leur tour, sécréter des anticorps anti-plaquettes cross-réactifs (pouvant non seulement reconnaître les plaquettes de rats mais également les plaquettes de souris) et (2) en augmentant, grâce à la sécrétion d’IFN-γ, la capacité des macrophages de phagocyter des plaquettes opsonisées. Enfin, nous avons démontré que des lymphocytes T CD4+CD25+ peuvent réguler l’induction et la rémission spontanée de la thrombopénie chez des souris immunisées. En effet, une thrombopénie plus sévère et de plus longue durée a été induite chez des souris immunisées dont les lymphocytes T régulateurs ont été déplétés à l’aide d’anticorps, contrairement à des souris chez qui les populations de lymphocytes T régulateurs étaient normales. Cependant, cette thrombopénie accrue n’était pas corrélée à une augmentation des anticorps à la surface des plaquettes. De façon similaire, l’administration de lymphocytes T régulateurs chez des souris naïves a eu un effet protecteur sur l’induction de thrombopénie lors d’immunisations ultérieures. Nous émettons l’hypothèse que la régulation de la réponse auto-immune est exercée au niveau des lymphocytes B bien que cette régulation pourrait également, en partie, être exercée au niveau des lymphocytes T.(SBIM 3) -- UCL, 201

Two-step mechanism of virus-induced autoimmune hemolytic anemia.

Viruses are associated with the development of autoantibody-mediated blood autoimmune diseases. A two-step mechanism could explain virus involvement in the development of experimental hemolytic anemia. Immunization of normal mice with rat erythrocytes results in an autoantibody production that could be enhanced by viral infection, without erythrocyte destruction. Inoculation of the same virus when autoantibodies are at high levels triggers clinical anemia. This results from macrophage activation by gamma-interferon, leading to exacerbated erythrophagocytosis. Thus the development of anemia during the course of viral infection may require two independent stimuli, in which the first triggers autoantibody production and the second enhances the pathogenicity of these autoantibodies

Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly individuals. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanized monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F protein with subnanomolar affinity. ALX-0171 demonstrated in vitro neutralization superior to that of palivizumab against prototypic RSV subtype A and B strains. Moreover, ALX-0171 completely blocked replication to below the limit of detection for 87% of the viruses tested, whereas palivizumab did so for 18% of the viruses tested at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease.This work was supported by the Agentschap voor Innovatie door Wetenschap en Techniek (IWT), Belgium (grant numbers 100333 and 130562). Work in Madrid was partially supported by grant SAF2012-31217 to J.A.M. from Plan Nacional I+D+i

Delivery of ALX-0171 by inhalation greatly reduces respiratory syncytial virus disease in newborn lambs

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory disease in infants and young children worldwide. Currently, treatment is supportive and no vaccines are available. The use of newborn lambs to model hRSV infection in human infants may provide a valuable tool to assess safety and efficacy of new antiviral drugs and vaccines. ALX-0171 is a trivalent Nanobody targeting the hRSV fusion (F) protein and its therapeutic potential was evaluated in newborn lambs infected with a human strain of RSV followed by daily ALX-0171 nebulization for 3 or 5 consecutive days. Colostrum-deprived newborn lambs were infected with hRSV-M37 before being treated by daily nebulization with either ALX-0171 or placebo. Two different treatment regimens were examined: day 1–5 or day 3–5 post-infection. Lambs were monitored daily for general well-being and clinical parameters. Respiratory tissues and bronchoalveolar lavage fluid were collected at day 6 post-inoculation for the quantification of viral lesions, lung viral titers, viral antigen and lung histopathology. Administration by inhalation of ALX-0171 was well-tolerated in these hRSV-infected newborn lambs. Robust antiviral effects and positive effects on hRSV-induced lung lesions and reduction in symptoms of illness were noted. These effects were still apparent when treatment start was delayed and coincided with peak viral loads (day 3 post-infection) and at a time point when signs of RSV disease were apparent. The latter design is expected to have high translational value for planned clinical trials. These results are indicative of the therapeutic potential of ALX-0171 in infants.This is the Version of Record of an article published by Taylor & Francis as Larios Mora, Alejandro, Laurent Detalle, Jack M. Gallup, Albert Van Geelen, Thomas Stohr, Linde Duprez, and Mark R. Ackermann. "Delivery of ALX-0171 by inhalation greatly reduces respiratory syncytial virus disease in newborn lambs." mAbs, vol. 10, no. 5, pp. 778-795. Taylor & Francis, 2018. DOI: 10.1080/19420862.2018.1470727. Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). Copyright 2018 Alejandro Larios Mora, Laurent Detalle, Jack M. Gallup, Albert Van Geelen, Thomas Stohr, Linde Duprez, and Mark R. Ackermann. Posted with permission

Enhancement of autoantibody pathogenicity by viral infections in mouse models of anemia and thrombocytopenia.

Viral infections are involved in the pathogenesis of blood autoimmune diseases such as hemolytic anemia and thrombocytopenia. Although antigenic mimicry has been proposed as a major mechanism by which viruses could trigger the development of such diseases, it is not easy to understand how widely different viruses might induce these blood autoimmune diseases by this sole mechanism. In mice infected with lactate dehydrogenase-elevating virus (LDV), or mouse hepatitis virus, and treated with anti-erythrocyte or anti-platelet monoclonal autoantibodies at a dose insufficient to induce clinical disease by themselves, the infection sharply enhances the pathogenicity of autoantibodies, leading to severe anemia or thrombocytopenia. This effect is observed only with antibodies that induce disease through phagocytosis. Moreover, the phagocytic activity of macrophages from infected mice is increased and the enhancing effect of infection on autoantibody-mediated pathogenicity is strongly suppressed by treatment of mice with clodronate-containing liposomes. Finally, the disease induced by LDV after administration of autoantibodies is largely suppressed in animals deficient for gamma-interferon receptor. Together, these observations suggest that viruses may trigger autoantibody-mediated anemia or thrombocytopenia by activating macrophages through gamma-interferon production, a mechanism that may account for the pathogenic similarities of multiple infectious agents

Kinetics of Respiratory Syncytial Virus (RSV) Memphis Strain 37 (M37) Infection in the Respiratory Tract of Newborn Lambs as an RSV Infection Model for Human Infants

Rationale Respiratory syncytial virus (RSV) infection in preterm and newborn infants can result in severe bronchiolitis and hospitalization. The lamb lung has several key features conducive to modeling RSV infection in human infants, including susceptibility to human strains of RSV such as the A2, Long, and Memphis Strain 37 (M37). In this study, the kinetics of M37 infection was investigated in newborn lambs in order to better define clinical, viral, physiological, and immunological parameters as well as the pathology and lesions. Methods Newborn lambs were nebulized with M37 hRSV (6 mL of 1.27 x 107 FFU/mL), monitored daily for clinical responses, and respiratory tissues were collected from groups of lambs at days 1, 3, 4, 6, and 8 post-inoculation for the assessment of viral replication parameters, lesions and also cellular, immunologic and inflammatory responses. Results Lambs had increased expiratory effort (forced expiration) at days 4, 6, and 8 post-inoculation. Nasal wash lacked RSV titers at day 1, but titers were present at low levels at days 3 (peak), 4, and 8. Viral titers in bronchoalveolar lavage fluid (BALF) reached a plateau at day 3 (4.6 Log10 FFU/mL), which was maintained until day 6 (4.83 Log10 FFU/mL), and were markedly reduced or absent at day 8. Viral RNA levels (detected by RT-qPCR) in BALF were indistinguishable at days 3 (6.22 ± 0.08 Log10 M37 RNA copies/mL; mean ± se) and 4 (6.20 ± 0.16 Log10 M37 RNA copies/mL; mean ± se) and increased slightly on day 6 (7.15 ± 0.2 Log10 M37 RNA copies/mL; mean ± se). Viral antigen in lung tissue as detected by immunohistochemistry was not seen at day 1, was present at days 3 and 4 before reaching a peak by day 6, and was markedly reduced by day 8. Viral antigen was mainly present in airways (bronchi, bronchioles) at day 3 and was increasingly present in alveolar cells at days 4 and 6, with reduction at day 8. Histopathologic lesions such as bronchitis/bronchiolitis, epithelial necrosis and hyperplasia, peribronchial lymphocyte infiltration, and syncytial cells, were consistent with those described previously for lambs and infants. Conclusion This work demonstrates that M37 hRSV replication in the lower airways of newborn lambs is robust with peak replication on day 3 and sustained until day 6. These findings, along with the similarities of lamb lung to those of infants in terms of alveolar development, airway branching and epithelium, susceptibility to human RSV strains, lesion characteristics (bronchiolitis), lung size, clinical parameters, and immunity, further establish the neonatal lamb as a model with key features that mimic RSV infection in infants.This article is from PLoS ONE 10(12): e0143580. doi:10.1371/journal.pone.0143580.</p

Kinetics of Respiratory Syncytial Virus (RSV) Memphis Strain 37 (M37) Infection in the Respiratory Tract of Newborn Lambs as an RSV Infection Model for Human Infants.

Respiratory syncytial virus (RSV) infection in preterm and newborn infants can result in severe bronchiolitis and hospitalization. The lamb lung has several key features conducive to modeling RSV infection in human infants, including susceptibility to human strains of RSV such as the A2, Long, and Memphis Strain 37 (M37). In this study, the kinetics of M37 infection was investigated in newborn lambs in order to better define clinical, viral, physiological, and immunological parameters as well as the pathology and lesions.Newborn lambs were nebulized with M37 hRSV (6 mL of 1.27 x 10(7) FFU/mL), monitored daily for clinical responses, and respiratory tissues were collected from groups of lambs at days 1, 3, 4, 6, and 8 post-inoculation for the assessment of viral replication parameters, lesions and also cellular, immunologic and inflammatory responses.Lambs had increased expiratory effort (forced expiration) at days 4, 6, and 8 post-inoculation. Nasal wash lacked RSV titers at day 1, but titers were present at low levels at days 3 (peak), 4, and 8. Viral titers in bronchoalveolar lavage fluid (BALF) reached a plateau at day 3 (4.6 Log10 FFU/mL), which was maintained until day 6 (4.83 Log10 FFU/mL), and were markedly reduced or absent at day 8. Viral RNA levels (detected by RT-qPCR) in BALF were indistinguishable at days 3 (6.22 ± 0.08 Log10 M37 RNA copies/mL; mean ± se) and 4 (6.20 ± 0.16 Log10 M37 RNA copies/mL; mean ± se) and increased slightly on day 6 (7.15 ± 0.2 Log10 M37 RNA copies/mL; mean ± se). Viral antigen in lung tissue as detected by immunohistochemistry was not seen at day 1, was present at days 3 and 4 before reaching a peak by day 6, and was markedly reduced by day 8. Viral antigen was mainly present in airways (bronchi, bronchioles) at day 3 and was increasingly present in alveolar cells at days 4 and 6, with reduction at day 8. Histopathologic lesions such as bronchitis/bronchiolitis, epithelial necrosis and hyperplasia, peribronchial lymphocyte infiltration, and syncytial cells, were consistent with those described previously for lambs and infants.This work demonstrates that M37 hRSV replication in the lower airways of newborn lambs is robust with peak replication on day 3 and sustained until day 6. These findings, along with the similarities of lamb lung to those of infants in terms of alveolar development, airway branching and epithelium, susceptibility to human RSV strains, lesion characteristics (bronchiolitis), lung size, clinical parameters, and immunity, further establish the neonatal lamb as a model with key features that mimic RSV infection in infants