SARS-CoV-2 spike protein induces monocyte apoptosis and interleukin-8 production

Background: In the setting of SARS-CoV-2 infection and COVID-19 illness, a subset of symptomatic patients has been reported to experience severe leukopenia. Viral proteins have been described to have the capacity to induce cell death in peripheral blood cells in infections such as HIV. Given the expression of the cognate receptor, ACE-2, on the surface of Peripheral blood mononuclear cells (PBMCs), we hypothesized that SARS-CoV-2 may induce leukopenia via spike protein ligand-receptor interaction. Methods: PBMCs were isolated from the fresh blood of normal donors and were treated with 1ug/ml of recombinant spike protein, and analyzed for cell death via the Incucyte Live/Cell imaging system. To measure subset specific cell death, PBMCs treated with recombinant spike protein for 48 hours were analyzed by flow cytometry for the expression of cell specific surface receptors and concomitant active caspase 3 expression. Culture supernatant was analyzed by multiplex cytokine analysis to evaluate the presence of pro-inflammatory cytokines. Similar assays were carried out in the presence of a spike- binding domain-antagonistic antibody in order to determine the specific role of spike- ACE2 interaction in causing cell death. Finally, cells from COVID positive patients were analyzed to determine if similar results were observable in-vivo. Results: The treatment of PBMCs with recombinant SARS-CoV-2 spike resulted in significant cell death over time in 2 out of three donors tested (p<0.05) by IncuCyte live imaging analysis. When analyzed for subset specific cell death, a significant increase in cell death (p<0.01), as measured by Caspase 3, was observed in CD14+CD3- cells, correlating with the monocyte population. Supernatants from these cultures demonstrated markedly increased IL-8 production (p=0.0536). Cultures carried out in the presence of a spike antagonistic antibody abrogated the effects of spike protein, indicating a direct relationship between spike-ACE2 interaction and cell death in this sub-population. Similar flow cytometric analysis from 5 febrile patients with COVID-19 demonstrated significantly increased monocyte apoptosis (p<0.05), compared to CD3+ lymphocytes from the same donors;whereas significantly increased monocyte apoptosis was not observed in 5 afebrile COVID-19 patients. Conclusion: These results indicate that SARS-CoV-2 spike protein may induce apoptosis specifically in Monocytes, in an ACE2 dependent manner, in some but not all patients.

GM-CSF Neutralization With Lenzilumab in Severe COVID-19 Pneumonia: A Case-Cohort Study.

OBJECTIVE: To assess the efficacy and safety of lenzilumab in patients with severe coronavirus disease 2019 (COVID-19) pneumonia. METHODS: Hospitalized patients with COVID-19 pneumonia and risk factors for poor outcomes were treated with lenzilumab 600 mg intravenously for three doses through an emergency single-use investigational new drug application. Patient characteristics, clinical and laboratory outcomes, and adverse events were recorded. We also identified a cohort of patients matched to the lenzilumab patients for age, sex, and disease severity. Study dates were March 13, 2020, to June 18, 2020. All patients were followed through hospital discharge or death. RESULTS: Twelve patients were treated with lenzilumab; 27 patients comprised the matched control cohort (untreated). Clinical improvement, defined as improvement of at least 2 points on the 8-point ordinal clinical endpoints scale, was observed in 11 of 12 (91.7%) patients treated with lenzilumab and 22 of 27 (81.5%) untreated patients. The time to clinical improvement was significantly shorter for the lenzilumab-treated group compared with the untreated cohort with a median of 5 days versus 11 days (P=.006). Similarly, the proportion of patients with acute respiratory distress syndrome (oxygen saturation/fraction of inspired oxygen<315 mm Hg) was significantly reduced over time when treated with lenzilumab compared with untreated (P<.001). Significant improvement in inflammatory markers (C-reactive protein and interleukin 6) and markers of disease severity (absolute lymphocyte count) were observed in patients who received lenzilumab, but not in untreated patients. Cytokine analysis showed a reduction in inflammatory myeloid cells 2 days after lenzilumab treatment. There were no treatment-emergent adverse events attributable to lenzilumab. CONCLUSION: In high-risk COVID-19 patients with severe pneumonia, granulocyte-macrophage colony-stimulating factor neutralization with lenzilumab was safe and associated with faster improvement in clinical outcomes, including oxygenation, and greater reductions in inflammatory markers compared with a matched control cohort of patients hospitalized with severe COVID-19 pneumonia. A randomized, placebo-controlled clinical trial to validate these findings is ongoing (NCT04351152).

Initial SARS-CoV-2 PCR crossing point does not predict hospitalization and duration of PCR positivity.

This study aimed to determine if the crossing point of the initial positive SARS-CoV-2 PCR test correlated with patient demographics, subsequent hospitalization, or duration of positivity. Seventy-three patients with two or more positive PCR tests had a median time of 23 days to two consecutive negative results.