Nonstructural Protein 1 of Variant PEDV Plays a Key Role in Escaping Replication Restriction by Complement C3.

Zoonotic coronaviruses represent an ongoing threat to public health. The classical porcine epidemic diarrhea virus (PEDV) first appeared in the early 1970s. Since 2010, outbreaks of highly virulent PEDV variants have caused great economic losses to the swine industry worldwide. However, the strategies by which PEDV variants escape host immune responses are not fully understood. Complement component 3 (C3) is considered a central component of the three complement activation pathways and plays a crucial role in preventing viral infection. In this study, we found that C3 significantly inhibited PEDV replication in vitro, and both variant and classical PEDV strains induced high levels of interleukin-1ß (IL-1ß) in Huh7 cells. However, the PEDV variant strain reduces C3 transcript and protein levels induced by IL-1ß compared with the PEDV classical strain. Examination of key molecules of the C3 transcriptional signaling pathway revealed that variant PEDV reduced C3 by inhibiting CCAAT/enhancer-binding protein ß (C/EBP-ß) phosphorylation. Mechanistically, PEDV nonstructural protein 1 (NSP1) inhibited C/EBP-ß phosphorylation via amino acid residue 50. Finally, we constructed recombinant PEDVs to verify the critical role of amino acid 50 of NSP1 in the regulation of C3 expression. In summary, we identified a novel antiviral role of C3 in inhibiting PEDV replication and the viral immune evasion strategies of PEDV variants. Our study reveals new information on PEDV-host interactions and furthers our understanding of the pathogenic mechanism of this virus. IMPORTANCE The complement system acts as a vital link between the innate and the adaptive immunity and has the ability to recognize and neutralize various pathogens. Activation of the complement system acts as a double-edged sword, as appropriate levels of activation protect against pathogenic infections, but excessive responses can provoke a dramatic inflammatory response and cause tissue damage, leading to pathological processes, which often appear in COVID-19 patients. However, how PEDV, as the most severe coronavirus causing diarrhea in piglets, regulates the complement system has not been previously reported. In this study, for the first time, we identified a novel mechanism of a PEDV variant in the suppression of C3 expression, showing that different coronaviruses and even different subtype strains differ in regulation of C3 expression. In addition, this study provides a deeper understanding of the mechanism of the PEDV variant in immune escape and enhanced virulence.

Structure and function of a family of tick-derived complement inhibitors targeting properdin.

Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches.

C5aR inhibition of nonimmune cells suppresses inflammation and maintains epithelial integrity in SARS-CoV-2-infected primary human airway epithelia.

BACKGROUND: Excessive inflammation triggered by a hitherto undescribed mechanism is a hallmark of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and is associated with enhanced pathogenicity and mortality. OBJECTIVE: Complement hyperactivation promotes lung injury and was observed in patients suffering from Middle East respiratory syndrome-related coronavirus, SARS-CoV-1, and SARS-CoV-2 infections. Therefore, we investigated the very first interactions of primary human airway epithelial cells on exposure to SARS-CoV-2 in terms of complement component 3 (C3)-mediated effects. METHODS: For this, we used highly differentiated primary human 3-dimensional tissue models infected with SARS-CoV-2 patient isolates. On infection, viral load, viral infectivity, intracellular complement activation, inflammatory mechanisms, and tissue destruction were analyzed by real-time RT-PCR, high content screening, plaque assays, luminex analyses, and transepithelial electrical resistance measurements. RESULTS: Here, we show that primary normal human bronchial and small airway epithelial cells respond to SARS-CoV-2 infection by an inflated local C3 mobilization. SARS-CoV-2 infection resulted in exaggerated intracellular complement activation and destruction of the epithelial integrity in monolayer cultures of primary human airway cells and highly differentiated, pseudostratified, mucus-producing, ciliated respiratory tissue models. SARS-CoV-2-infected 3-dimensional cultures secreted significantly higher levels of C3a and the proinflammatory cytokines IL-6, monocyte chemoattractant protein 1, IL-1α, and RANTES. CONCLUSIONS: Crucially, we illustrate here for the first time that targeting the anaphylotoxin receptors C3a receptor and C5a receptor in nonimmune respiratory cells can prevent intrinsic lung inflammation and tissue damage. This opens up the exciting possibility in the treatment of COVID-19.

ColdZyme Maintains Integrity in SARS-CoV-2-Infected Airway Epithelia.

SARS-CoV-2 infection causing the COVID-19 pandemic calls for immediate interventions to avoid viral transmission, disease progression, and subsequent excessive inflammation and tissue destruction. Primary normal human bronchial epithelial cells are among the first targets of SARS-CoV-2 infection. Here, we show that ColdZyme medical device mouth spray efficiently protected against virus entry, excessive inflammation, and tissue damage. Applying ColdZyme to fully differentiated, polarized human epithelium cultured at an air-liquid interphase (ALI) completely blocked binding of SARS-CoV-2 and increased local complement activation mediated by the virus as well as productive infection of the tissue model. While SARS-CoV-2 infection resulted in exaggerated intracellular complement activation immediately following infection and a drop in transepithelial resistance, these parameters were bypassed by single pretreatment of the tissues with ColdZyme mouth spray. Crucially, our study highlights the importance of testing already evaluated and safe drugs such as ColdZyme mouth spray for maintaining epithelial integrity and hindering SARS-CoV-2 entry within standardized three-dimensional (3D) in vitro models mimicking the in vivo human airway epithelium.IMPORTANCE Although our understanding of COVID-19 continuously progresses, essential questions regarding prophylaxis and treatment remain open. A hallmark of severe SARS-CoV-2 infection is a hitherto-undescribed mechanism leading to excessive inflammation and tissue destruction associated with enhanced pathogenicity and mortality. To tackle the problem at the source, transfer of SARS-CoV-2, subsequent binding, infection, and inflammatory responses have to be avoided. In this study, we used fully differentiated, mucus-producing, and ciliated human airway epithelial cultures to test the efficacy of ColdZyme medical device mouth spray in terms of protection from SARS-CoV-2 infection. Importantly, we found that pretreatment of the in vitro airway cultures using ColdZyme mouth spray resulted in significantly shielding the epithelial integrity, hindering virus binding and infection, and blocking excessive intrinsic complement activation within the airway cultures. Our in vitro data suggest that ColdZyme mouth spray may have an impact in prevention of COVID-19.

A double edged-sword - The Complement System during SARS-CoV-2 infection.

In the past 20 years, infections caused by coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 have posed a threat to public health since they may cause severe acute respiratory syndrome (SARS) in humans. The Complement System is activated during viral infection, being a central protagonist of innate and acquired immunity. Here, we report some interactions between these three coronaviruses and the Complement System, highlighting the central role of C3 with the severity of these infections. Although it can be protective, its role during coronavirus infections seems to be contradictory. For example, during SARS-CoV-2 infection, Complement System can control the viral infection in asymptomatic or mild cases; however, it can also intensify local and systemic damage in some of severe COVID-19 patients, due to its potent proinflammatory effect. In this last condition, the activation of the Complement System also amplifies the cytokine storm and the pathogenicity of coronavirus infection. Experimental treatment with Complement inhibitors has been an enthusiastic field of intense investigation in search of a promising additional therapy in severe COVID-19 patients.

Complement C3 vs C5 inhibition in severe COVID-19: Early clinical findings reveal differential biological efficacy.

Growing clinical evidence has implicated complement as a pivotal driver of COVID-19 immunopathology. Deregulated complement activation may fuel cytokine-driven hyper-inflammation, thrombotic microangiopathy and NET-driven immunothrombosis, thereby leading to multi-organ failure. Complement therapeutics have gained traction as candidate drugs for countering the detrimental consequences of SARS-CoV-2 infection. Whether blockade of terminal complement effectors (C5, C5a, or C5aR1) may elicit similar outcomes to upstream intervention at the level of C3 remains debated. Here we compare the efficacy of the C5-targeting monoclonal antibody eculizumab with that of the compstatin-based C3-targeted drug candidate AMY-101 in small independent cohorts of severe COVID-19 patients. Our exploratory study indicates that therapeutic complement inhibition abrogates COVID-19 hyper-inflammation. Both C3 and C5 inhibitors elicit a robust anti-inflammatory response, reflected by a steep decline in C-reactive protein and IL-6 levels, marked lung function improvement, and resolution of SARS-CoV-2-associated acute respiratory distress syndrome (ARDS). C3 inhibition afforded broader therapeutic control in COVID-19 patients by attenuating both C3a and sC5b-9 generation and preventing FB consumption. This broader inhibitory profile was associated with a more robust decline of neutrophil counts, attenuated neutrophil extracellular trap (NET) release, faster serum LDH decline, and more prominent lymphocyte recovery. These early clinical results offer important insights into the differential mechanistic basis and underlying biology of C3 and C5 inhibition in COVID-19 and point to a broader pathogenic involvement of C3-mediated pathways in thromboinflammation. They also support the evaluation of these complement-targeting agents as COVID-19 therapeutics in large prospective trials.

Concomitant new diagnosis of systemic lupus erythematosus and COVID-19 with possible antiphospholipid syndrome. Just a coincidence? A case report and review of intertwining pathophysiology.

In the midst of the COVID-19 pandemic, further understanding of its complications points towards dysregulated immune response as a major component. Systemic lupus erythematosus (SLE) is also a disease of immune dysregulation leading to multisystem compromise. We present a case of new-onset SLE concomitantly with COVID-19 and development of antiphospholipid antibodies. An 18-year-old female that presented with hemodynamic collapse and respiratory failure, progressed to cardiac arrest, and had a pericardial tamponade drained. She then progressed to severe acute respiratory distress syndrome, severe ventricular dysfunction, and worsening renal function with proteinuria and hematuria. Further studies showed bilateral pleural effusions, positive antinuclear and antidouble-stranded DNA antibodies, lupus anticoagulant, and anticardiolipin B. C3 and C4 levels were low. SARS-Cov-2 PCR was positive after 2 negative tests. She also developed multiple deep venous thrombosis, in the setting of positive antiphospholipid antibodies and lupus anticoagulant. In terms of pathophysiology, COVID-19 is believed to cause a dysregulated cytokine response which could potentially be exacerbated by the shift in Th1 to Th2 response seen in SLE. Also, it is well documented that viral infections are an environmental factor that contributes to the development of autoimmunity; however, COVID-19 is a new entity, and it is not known if it could trigger autoimmune conditions. Additionally, it is possible that SARS-CoV-2, as it happens with other viruses, might lead to the formation of antiphospholipid antibodies, potentially contributing to the increased rates of thrombosis seen in COVID-19.

COVID-19 and Acute Lupus Pneumonitis: Diagnostic and Treatment Dilemma.

In this article, we present a case of a young female patient with previously diagnosed lupus pneumonitis, now with a flare and new superimposed COVID-19 infection that was treated with intravenous steroids. On computed tomography scans, she had extensive interstitial lung fibrosis in addition to a positive COVID-19 polymerase chain reaction test requiring 6 L of oxygen via nasal cannula on admission. After administration of methylprednisolone, the patient improved and was weaned off her oxygen requirements and was discharged home.