Severe COVID-19 patients display hyper-activated NK cells and NK cell-platelet aggregates.

COVID-19 is characterised by a broad spectrum of clinical and pathological features. Natural killer (NK) cells play an important role in innate immune responses to viral infections. Here, we analysed the phenotype and activity of NK cells in the blood of COVID-19 patients using flow cytometry, single-cell RNA-sequencing (scRNA-seq), and a cytotoxic killing assay. In the plasma of patients, we quantified the main cytokines and chemokines. Our cohort comprises COVID-19 patients hospitalised in a low-care ward unit (WARD), patients with severe COVID-19 disease symptoms hospitalised in intensive care units (ICU), and post-COVID-19 patients, who were discharged from hospital six weeks earlier. NK cells from hospitalised COVID-19 patients displayed an activated phenotype with substantial differences between WARD and ICU patients and the timing when samples were taken post-onset of symptoms. While NK cells from COVID-19 patients at an early stage of infection showed increased expression of the cytotoxic molecules perforin and granzyme A and B, NK cells from patients at later stages of COVID-19 presented enhanced levels of IFN-γ and TNF-α which were measured ex vivo in the absence of usual in vitro stimulation. These activated NK cells were phenotyped as CD49a+CD69a+CD107a+ cells, and their emergence in patients correlated to the number of neutrophils, and plasma IL-15, a key cytokine in NK cell activation. Despite lower amounts of cytotoxic molecules in NK cells of patients with severe symptoms, majority of COVID-19 patients displayed a normal cytotoxic killing of Raji tumour target cells. In vitro stimulation of patients blood cells by IL-12+IL-18 revealed a defective IFN-γ production in NK cells of ICU patients only, indicative of an exhausted phenotype. ScRNA-seq revealed, predominantly in patients with severe COVID-19 disease symptoms, the emergence of an NK cell subset with a platelet gene signature that we identified by flow and imaging cytometry as aggregates of NK cells with CD42a+CD62P+ activated platelets. Post-COVID-19 patients show slow recovery of NK cell frequencies and phenotype. Our study points to substantial changes in NK cell phenotype during COVID-19 disease and forms a basis to explore the contribution of platelet-NK cell aggregates to antiviral immunity against SARS-CoV-2 and disease pathology.

Live-attenuated YF17D-vectored COVID-19 vaccine protects from lethal yellow fever virus infection in mouse and hamster models.

BACKGROUND: The live-attenuated yellow fever vaccine YF17D holds great promise as alternative viral vector vaccine platform, showcased by our previously presented potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidate YF-S0. Besides protection from SARS-CoV-2, YF-S0 also induced strong yellow fever virus (YFV)-specific immunity, suggestive for full dual activity. A vaccine concomitantly protecting from SARS-CoV-2 and YFV would be of great benefit for those living in YFV-endemic areas with limited access to current SARS-CoV-2 vaccines. However, for broader applicability, pre-existing vector immunity should not impact the potency of such YF17D-vectored vaccines. METHODS: The immunogenicity and efficacy of YF-S0 against YFV and SARS-CoV-2 in the presence of strong pre-existing YFV immunity were evaluated in mouse and hamster challenge models. FINDINGS: Here, we show that a single dose of YF-S0 is sufficient to induce strong humoral and cellular immunity against YFV as well as SARS-CoV-2 in mice and hamsters; resulting in full protection from vigorous YFV challenge in either model; in mice against lethal intracranial YF17D challenge, and in hamsters against viscerotropic infection and liver disease following challenge with highly pathogenic hamster-adapted YFV-Asibi strain. Importantly, strong pre-existing immunity against the YF17D vector did not interfere with subsequent YF-S0 vaccination in mice or hamsters; nor with protection conferred against SARS-CoV-2 strain B1.1.7 (Alpha variant) infection in hamsters. INTERPRETATION: Our findings warrant the development of YF-S0 as dual SARS-CoV-2 and YFV vaccine. Contrary to other viral vaccine platforms, use of YF17D does not suffer from pre-existing vector immunity. FUNDING: Stated in the acknowledgments.

Kinetics of peripheral blood neutrophils in severe coronavirus disease 2019.

OBJECTIVES: Emerging evidence of dysregulation of the myeloid cell compartment urges investigations on neutrophil characteristics in coronavirus disease 2019 (COVID-19). We isolated neutrophils from the blood of COVID-19 patients receiving general ward care and from patients hospitalised at intensive care units (ICUs) to explore the kinetics of circulating neutrophils and factors important for neutrophil migration and activation. METHODS: Multicolour flow cytometry was exploited for the analysis of neutrophil differentiation and activation markers. Multiplex and ELISA technologies were used for the quantification of protease, protease inhibitor, chemokine and cytokine concentrations in plasma. Neutrophil polarisation responses were evaluated microscopically. Gelatinolytic and metalloproteinase activity in plasma was determined using a fluorogenic substrate. Co-culturing healthy donor neutrophils with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) allowed us to investigate viral replication in neutrophils. RESULTS: Upon ICU admission, patients displayed high plasma concentrations of granulocyte-colony-stimulating factor (G-CSF) and the chemokine CXCL8, accompanied by emergency myelopoiesis as illustrated by high levels of circulating CD10-, immature neutrophils with reduced CXCR2 and C5aR expression. Neutrophil elastase and non-metalloproteinase-derived gelatinolytic activity were increased in plasma from ICU patients. Significantly higher levels of circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) in patients at ICU admission yielded decreased total MMP proteolytic activity in blood. COVID-19 neutrophils were hyper-responsive to CXCL8 and CXCL12 in shape change assays. Finally, SARS-CoV-2 failed to replicate inside human neutrophils. CONCLUSION: Our study provides detailed insights into the kinetics of neutrophil phenotype and function in severe COVID-19 patients, and supports the concept of an increased neutrophil activation state in the circulation.

Atypical response to bacterial coinfection and persistent neutrophilic bronchoalveolar inflammation distinguish critical COVID-19 from influenza.

Neutrophils are recognized as important circulating effector cells in the pathophysiology of severe coronavirus disease 2019 (COVID-19). However, their role within the inflamed lungs is incompletely understood. Here, we collected bronchoalveolar lavage (BAL) fluids and parallel blood samples of critically ill COVID-19 patients requiring invasive mechanical ventilation and compared BAL fluid parameters with those of mechanically ventilated patients with influenza, as a non-COVID-19 viral pneumonia cohort. Compared with those of patients with influenza, BAL fluids of patients with COVID-19 contained increased numbers of hyperactivated degranulating neutrophils and elevated concentrations of the cytokines IL-1ß, IL-1RA, IL-17A, TNF-α, and G-CSF; the chemokines CCL7, CXCL1, CXCL8, CXCL11, and CXCL12α; and the protease inhibitors elafin, secretory leukocyte protease inhibitor, and tissue inhibitor of metalloproteinases 1. In contrast, α-1 antitrypsin levels and net proteolytic activity were comparable in COVID-19 and influenza BAL fluids. During antibiotic treatment for bacterial coinfections, increased BAL fluid levels of several activating and chemotactic factors for monocytes, lymphocytes, and NK cells were detected in patients with COVID-19 whereas concentrations tended to decrease in patients with influenza, highlighting the persistent immunological response to coinfections in COVID-19. Finally, the high proteolytic activity in COVID-19 lungs suggests considering protease inhibitors as a treatment option.

Discriminating mild from critical COVID-19 by innate and adaptive immune single-cell profiling of bronchoalveolar lavages.

How the innate and adaptive host immune system miscommunicate to worsen COVID-19 immunopathology has not been fully elucidated. Here, we perform single-cell deep-immune profiling of bronchoalveolar lavage (BAL) samples from 5 patients with mild and 26 with critical COVID-19 in comparison to BALs from non-COVID-19 pneumonia and normal lung. We use pseudotime inference to build T-cell and monocyte-to-macrophage trajectories and model gene expression changes along them. In mild COVID-19, CD8+ resident-memory (TRM) and CD4+ T-helper-17 (TH17) cells undergo active (presumably antigen-driven) expansion towards the end of the trajectory, and are characterized by good effector functions, while in critical COVID-19 they remain more naïve. Vice versa, CD4+ T-cells with T-helper-1 characteristics (TH1-like) and CD8+ T-cells expressing exhaustion markers (TEX-like) are enriched halfway their trajectories in mild COVID-19, where they also exhibit good effector functions, while in critical COVID-19 they show evidence of inflammation-associated stress at the end of their trajectories. Monocyte-to-macrophage trajectories show that chronic hyperinflammatory monocytes are enriched in critical COVID-19, while alveolar macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, are depleted. In critical COVID-19, monocytes contribute to an ATP-purinergic signaling-inflammasome footprint that could enable COVID-19 associated fibrosis and worsen disease-severity. Finally, viral RNA-tracking reveals infected lung epithelial cells, and a significant proportion of neutrophils and macrophages that are involved in viral clearance.

A single-dose live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate.

The expanding pandemic of coronavirus disease 2019 (COVID-19) requires the development of safe, efficacious and fast-acting vaccines. Several vaccine platforms are being leveraged for a rapid emergency response1. Here we describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. We assess vaccine safety, immunogenicity and efficacy in several animal models. YF-S0 has an excellent safety profile and induces high levels of SARS-CoV-2 neutralizing antibodies in hamsters (Mesocricetus auratus), mice (Mus musculus) and cynomolgus macaques (Macaca fascicularis), and-concomitantly-protective immunity against yellow fever virus. Humoral immunity is complemented by a cellular immune response with favourable T helper 1 polarization, as profiled in mice. In a hamster model2 and in macaques, YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose conferred protection from lung disease in most of the vaccinated hamsters within as little as 10 days. Taken together, the quality of the immune responses triggered and the rapid kinetics by which protective immunity can be attained after a single dose warrant further development of this potent SARS-CoV-2 vaccine candidate.