In a recent study published on the bioRxiv * prepress server, researchers examined how the open-loop protein 8 (ORF8) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induced cell interaction. Host dendritic cells (DC). cytokine storm.
Study: The unique ORS8 protein of SARS-CoV-2 binds to human dendritic cells and induces a hyperinflammatory cytokine storm. Image Credit: NIAID
Fund
DCs are host innate immune system antigen presenting cells that infiltrate the human lungs after a pathogenic infection and differentiate into monocyte-derived DCs (moDCs) to help eliminate viral infections. including coronavirus disease 2019 (COVID-19). DCs activate T and B cells to suppress the progression of the disease, the failure of which could lead to a second innate immune response characterized by the rapid onset of widespread inflammation, often called a cytokine storm. In particular, the cytokine storm drives the severity and magnitude of acute respiratory distress syndrome (ARDS), the most common cause of death in patients with COVID-19.
The SARS-CoV-2 ORF8 gene is different from the SARS-CoV ORF8 gene and the Middle East Respiratory Syndrome (MERS-CoV) coronavirus. Studies have shown its value as a marker of early diagnosis of SARS-CoV-2 infection. However, studies demonstrating function and interactions between DC and ORF8 are scarce.
About the study
In the present study, the researchers aimed to study the DCs-ORF8 interaction and its contribution to the cytokine storm observed in patients with COVID-19. They isolated purified ORF8 from HEK293 cells transfected with ORF8 plasmid. They aimed to define the function of ORF8 as an immune modulator and virulence factor and analyzed whether ORF8 could influence the process of differentiating DCs.
In addition, they investigated whether ORF8 directly activated monocyte differentiation in DC. To this end, the team differentiated monocytes from moDC with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) in the presence or absence of ORF8 at different concentrations. They increased the dose of ORF8 protein to investigate the positive regulation of DC maturation markers, differentiation cluster 40 (CD40), and CD80.
In addition, the team used immune precipitation (IP) to investigate the ORF8 interaction with the DC marker, DC-SIGN. For multiple cytokine analysis, the researchers collected DC supernatants differentiated from several donors in the presence or absence of ORF8 protein. They also analyzed the fingerprints of cytokines and chemokines of DCs exposed to ORF8 during the DC differentiation process.
The team performed ribonucleic acid (RNA) sequencing to further characterize the proinflammatory response of ORF8-treated DCs. They investigated the ability of neutralizing antibodies in patient serums to neutralize the ORF8-induced effect. Finally, the team performed an enzyme-linked immunosorbent assay (ELISA) tailored to detect ORF8 antibodies in the convalescent plasma of patients with COVID-19.
Crystal structure of SARS-CoV-2 ORF8 and related CoV homologues. a) Crystal structure of the SARS-CoV-2 ORF8 dimer. The sequence recognized by α-ORF8 antibodies (residues 41-90) (83) is highlighted in red and indicated. Intra and intermolecular disulfide bridges are shown as poles, and regions with missing electron density (residues 63-77) are shown as dashed lines. b) Overlay of the ORF8 structure determined here (gray), and the previously deposited 7JTL structure (cyan). The A (transparent) chain of 7JTL and 7MX9 was aligned to show the change in the relative orientation of the B chain within the dimer. The surface representations highlight the closed shape of the 7MX9 relative to the 7JTL. c) Individual monomers of 7MX9 (black), 7JTL (cyan) and 7JX6 (magenta) are superimposed and the loop regions between β2-β3, β3-β4, β4-β5 and β7-β8 are indicated by dotted circles. Only Cα atoms are shown for clarity; d) Overlay of OrV8 CoV homology models. ORF8 counterparts of RaTG13-CoV bat (red), CoV bat type Rs3367 (green) and SARS-CoV-2 (blue), superimposed on the SARS-CoV-2 ORF8 template (7JTL; yellow). Part of the specific loop region of ORF8 (residues 63-77) is highlighted in gray. Disordered regions of N terminals have been removed and a single ORF8 monomer has been shown for clarity.
Study results
Extracellular or secreted ORF8 protein specifically binds DCs and their parental monocytes. A α-ORF8 polyclonal antibody reduced its binding, demonstrating the specificity of this binding interaction. ORF8 alone could not trigger DC differentiation, as precursor cells remained in a CD14 + monocyte stage. Serum bovine serum albumin (BSA) or ORF8 monocyte precursor cells remained positive for the CD14 monocyte marker and negative for DC-SIGN and CD1c. Accordingly, the authors noted the induction of DC maturation depending on the ORF8 dose.
In addition, cells treated with ORF8 showed a significantly different stellate morphology comparable to mature DCs. In addition, the authors noted a positive regulation of maturation markers, including the major histocompatibility complex II (MHCII), CD83, CD80, CD86, and CD40 in ORF8-treated cells. In contrast, DC’s CD11c marker decreased slightly.
ORF8 did not affect monocytes, similar to BSA, and did not act as an inducer of DC differentiation. Surprisingly, ORF8-treated cells had elevated levels of interferon gamma-induced protein (IF-γ) (IP-10), interleukin 1-beta (IL-1β), tumor necrosis factor-alpha (TNF-α). , IFN- γ and IL-8. The effect could be partially reversed by the simultaneous addition of a polyclonal rabbit antibody neutralizing α-ORF8 against ORF8 during differentiation, verifying the specific induction of ORF8 by the cytokine storm.
A co-IP assay showed that ORF8 interacted with the DC-SIGN receiver. In addition, an anti-ORF8 antibody interfered with the DC-SIGN interaction side.
RNA sequencing data further confirmed that ORF8 played a role in the progression and course of the COVID-19 cytokine storm activating DCs. Consequently, the authors noted 211 RNAs unique to ORF8 in addition to 632 RNAs that reflected inflammatory overlaps between lipopolysaccharide (LPS), a positive control, and ORF8. The authors found eight of the 64 patients infected with SARS-CoV-2 highly positive for anti-ORF8 IgG antibodies, which had the potential to neutralize the ORF8 effect on DCs.
ORF8 induces an inflammatory mRNA profile involved in SARS-CoV-2 infection a) 1) Enriched pathway network design where each term is represented by a circular node, where its size is proportional to the number of genes of input that fall into this term, and its color represents its cluster identity (that is, nodes of the same color belong to the same cluster). Terms with a similarity score> 0.3 are linked by a border (the thickness of the border represents the similarity score). The network is visualized with Cytoscape (v3.1.2) with a “force-driven” design and the grouped edge for clarity. 2) The same enrichment network has its nodes displayed as cakes. Each cake sector is proportional to the number of visits originating from the list of genes analyzed. The color code of the cake sector represents the number of genes in each list and is consistent with the colors in the legend in the figure www.metascape.org/COVID. b) Comparison of the Venn diagram of the genes detected in the ORF8 data set compared to the published data sets (genes related to host antivirals / host cytokine genes; genes in APC).
Conclusions
Overall, the results of the study showed how the ORF8 protein interacted with DCs to cause a cytokine storm that caused ARDS in some severe COVID-19 cases. Therefore, preventing the onset of the ORF-8-mediated cytokine and chemokine response could lead to new corrective therapy for severe COVID-19. Current treatments focus on individual cytokines or the neutralizing SARS-CoV-2 ear protein; however, the study showed that neutralizing ORF8 protein could be a more promising approach to prevent progression to severe COVID-19.
Antibodies to ORF8 were unable to neutralize the immunomodulatory function of ORF8 and even improved binding to DCs, a phenomenon called antibody-dependent enhancement or ADE. Studies have documented ADE during infection by several viruses, including influenza and human immunodeficiency virus-1 (HIV-1). In the context of SARS-CoV-2, multiple studies have shown a high antibody response against the ORF8 protein. These antibodies could bind to the fraction, crystallizable receptors (FcR) of DCs, giving rise to ADE. These findings could help cause antibodies to neutralize ORF8 or block FcR that could mitigate the effect of OFR8 on DCs.
* Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guided by clinical practice / health-related behavior, or treated as established information.
Magazine reference:
- SARS-CoV-2’s unique ORF8 protein binds to human dendritic cells and induces a hyperinflammatory cytokine storm Matthias Hamdorf, Thomas Imhof, Ben Allan Bailey-Elkin, Janina Betz, Sebastian J Theobald, Alexander Simonis, Veronica Di Cristanziano, Lutz. Gieselmann, Felix Dewald, Clara Lehmann, Max Augustin, Florian Klein, Miguel Alejandre Alcazar, Robert Rogisch, Mario Fabri, Jan Rybniker, Heike Goebel, Jörg Stetefeld, Bent Brachvogel, Claus Cursiefen, Manuel Koch, Felix Bock, bioRxiv preprint 2022, DOI :