ABSTRACT
The open reading frame 8 (ORF8) protein, encoded by the SARS-CoV-2 virus after infection, stimulates monocytes/macrophages to produce pro-inflammatory cytokines. We hypothesized that a positive ex vivo monocyte response to ORF8 protein pre-COVID-19 would be associated with subsequent severe COVID-19. We tested ORF8 ex vivo on peripheral blood mononuclear cells (PBMCs) from 26 anonymous healthy blood donors and measured intracellular cytokine/chemokine levels in monocytes by flow cytometry. The % monocytes staining positive in the sample and change in mean fluorescence intensity (ΔMFI) after ORF8 were used to calculate the adjusted MFI for each cytokine. We then tested pre-COVID-19 PBMC samples from 60 CLL patients who subsequently developed COVID-19 infection. Severe COVID-19 was defined as hospitalization due to COVID-19. In the 26 normal donor samples, the adjusted MFI for interleukin (IL)-1ß, IL-6, IL-8, and CCL-2 were significantly different with ORF8 stimulation vs controls. We next analyzed monocytes from pre-COVID-19 PBMC samples from 60 CLL patients. The adjusted MFI to ORF8 stimulation of monocyte intracellular IL-1ß was associated with severe COVID-19 and a reactive ORF8 monocyte response was defined as an IL- 1ß adjusted MFI ≥ 0.18 (sensitivity 67%, specificity 75%). The median time to hospitalization after infection in CLL patients with a reactive ORF8 response was 12 days versus not reached for patients with a non-reactive ORF8 response with a hazard ratio of 7.7 (95% CI: 2.4-132, p=0.005). These results provide new insight on the monocyte inflammatory response to virus with implications in a broad range of disorders involving monocytes.
ABSTRACT
Despite extensive research, the specific factor associated with SARS-CoV-2 infection that mediates the life-threatening inflammatory cytokine response in patients with severe COVID-19 remains unidentified. Herein we demonstrate that the virus-encoded Open Reading Frame 8 (ORF8) protein is abundantly secreted as a glycoprotein in vitro and in symptomatic patients with COVID-19. ORF8 specifically binds to the NOD-like receptor family pyrin domain-containing 3 (NLRP3) in CD14+ monocytes to induce inflammasomal cytokine/chemokine responses including IL1ß, IL8, and CCL2. Levels of ORF8 protein in the blood correlate with severity and disease-specific mortality in patients with acute SARS-CoV-2 infection. Furthermore, the ORF8-induced inflammasome response was readily inhibited by the NLRP3 inhibitor MCC950 in vitro. Our study identifies a dominant cause of pathogenesis, its underlying mechanism, and a potential new treatment strategy for severe COVID-19.
ABSTRACT
Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post-translational modifications in the protein's native environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real-time monitoring of protein expression, and also provides an affinity handle for one-step protein purification using a suitable affinity reagent. Here, we describe a panel of anti-GFP and anti-mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target proteins using X-ray crystallography. From these analyses, we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody-sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry-tag system by using it to purify recombinant human topoisomerase 2α expressed in HEK293F cells. The mCherry-tag and GFP/YFP-tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real-time monitoring of protein expression levels and a high-efficiency purification step is needed.
