In a recent study published on the bioRxiv * prepress server, researchers investigated how the open-reading frame protein 8 (ORF8) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with host dendritic cells (DCs). induces a cytokine storm.
Study: The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and causes a hyperinflammatory cytokine storm. Image credit: NIAID
Background
DCs are antigen-presenting cells of the host’s innate immune system that infiltrate the human lung after pathogenic infection and differentiate into monocyte-derived DCs (moDCs) to help clear viral infections, including coronavirus disease 2019 (COVID). -19). DC activates T and B cells to suppress disease progression, the failure of which can 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 leads to the severity and extent of acute respiratory distress syndrome (ARDS), the most common cause of death in patients with COVID-19.
The ORF8 gene for SARS-CoV-2 is different from the ORF8 gene for SARS-CoV and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Studies show its value as an early diagnostic marker for SARS-CoV-2 infection. However, studies demonstrating the function and interactions between DC and ORF8 are scarce.
About the study
In the present study, the researchers aimed to study the interaction of DCs-ORF8 and its contribution to the cytokine storm observed in patients with COVID-19. They isolated purified ORF8 from HEK293 cells transfected with plasmid ORF8. They aimed to define the function of ORF8 as an immune modulator and virulence factor and analyzed whether ORF8 can affect the process of differentiation of DCs.
In addition, they investigated whether ORF8 directly triggered the differentiation of monocytes to DC. To this end, the team differentiated monocytes into moDCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) in the presence or absence of ORF8 at various concentrations. They increased the dose of ORF8 protein to investigate the increase in the regulation of DC maturation markers, cluster of differentiation 40 (CD40) and CD80.
The team also used immune precipitation (IP) to study the interaction of ORF8 with the DC marker, DC-SIGN. For multiplex cytokine analysis, the researchers collected the differentiated DC supernatant from several donors in the presence or absence of ORF8 protein. They also analyzed the cytokine and chemokine fingerprints of DCs exposed to ORF8 during the DC differentiation process.
The team performed ribonucleic acid (RNA) sequencing to further characterize the pro-inflammatory response of the ORF8 DC-treated. They investigated the ability of neutralizing antibodies in patients’ sera to neutralize the ORF8-induced effect. Finally, the team performed a specially performed enzyme-linked immunosorbent assay (ELISA) to detect ORF8 antibodies in the convalescent plasma of patients with COVID-19.
Crystal structure of SARS-CoV-2 ORF8 and related homologues of CoV. a) Crystal structure of the SARS-CoV-2 ORF8 dimer. The sequence recognized by α-ORF8 antibodies (residues 41-90) (83) is marked in red and labeled. Intra- and intermolecular disulfide bridges are shown as rods, and areas with missing electron density (residues 63-77) are shown as dotted lines. b) Superposition of the ORF8 structure defined herein (gray) and the pre-deposited 7JTL structure (cyan). Chain A (transparent) of 7JTL and 7MX9 were aligned to show the change in the relative orientation of chain B in the dimer. Surface images emphasize the closed shape of the 7MX9 compared to 7JTL. (c) Individual monomers of 7MX9 (black), 7JTL (cyan) and 7JX6 (purple) are superimposed and the areas of the loops between β2-β3, β3-β4, β4-β5 and β7-β8 are indicated by dotted circles. For greater clarity, only Cα atoms are shown; d) Superposition of homologous models of CoV ORF8. Bat homologues-CoV RaTG13 (red), bat-like CoV Rs3367 (green) and SARS-CoV-2 (blue) ORF8 superimposed on the SARS-CoV-2 ORF8 template (7JTL; yellow). Part of the ORF8-specific chain (residues 63-77) is highlighted in gray. The broken areas at the N-terminus were removed and a single ORF8 monomer was shown for clarity.
Survey results
The extracellular or secreted ORF8 protein specifically binds DCs and their parent monocytes. Polyclonal α-ORF8 antibody reduces its binding, demonstrating the specificity of this binding interaction. ORF8 alone cannot induce DC differentiation, as precursor cells remain in the CD14 + monocyte stage. Monocyte precursor cells treated with bovine serum albumin (BSA) or ORF8 remained positive for the monocyte marker CD14 and negative for DC-SIGN and CD1c. Accordingly, the authors noted ORF8 dose-dependent induction of DC maturation.
In addition, ORF8-treated cells showed significantly different star morphology comparable to mature DCs. In addition, the authors noted increased regulation of maturation markers, including major histocompatibility complex II (MHCII), CD83, CD80, CD86, and CD40 in ORF8-treated cells. In contrast, the DC marker CD11c was slightly lowered.
ORF8 does not affect monocytes, like BSA, and does not act as an inducer of DC differentiation. Surprisingly, ORF8-treated cells have elevated levels of interferon-gamma (IF-γ) -induced protein (IP-10), interleukin 1-beta (IL-1β), tumor necrosis factor-alpha (TNF-α), IFN-γ and IL-8. The effect can be partially reversed by the simultaneous addition of a neutralizing polyclonal rabbit antibody α-ORF8 against ORF8 during differentiation, confirming the ORF8-specific induction of the cytokine storm.
Co-IP analysis showed that ORF8 interacts with the DC-SIGN receptor. In addition, the anti-ORF8 antibody interferes with the DC-SIGN interaction side.
RNA sequencing data further confirm that ORF8 plays a role in the progression and course of the cytokine storm of COVID-19 by activating DCs. Therefore, the authors noted 211 unique RNAs for ORF8, in addition to 632 RNAs that reflect inflammatory overlaps between lipopolysaccharide (LPS), positive control, and ORF8. The authors found eight of 64 patients infected with SARS-CoV-2, highly positive for anti-ORF8 IgG antibodies, which have the potential to neutralize the effect of ORF8 on DCs.
ORF8 induces an inflammatory profile of mRNA involved in SARS-CoV-2 infection a) 1) Enriched pathway network layout, where each term is represented by a circular node, where its size is proportional to the number of input genes falling into this term, and its color represents its cluster identity (ie, nodes of the same color belong to the same cluster). Terms with a similarity score> 0.3 are related to an edge (the thickness of the edge represents the similarity score). The web is visualized with Cytoscape (v3.1.2) with a “force-oriented” layout and combined edges for clarity. 2) The same enrichment network has its nodes shown as pies. Each pie sector is proportional to the number of hits derived from the analyzed gene list. The color code for the pie sector represents the number of genes in each list and is consistent with the colors of the legend in the figure www.metascape.org/COVID. b) Comparison of Venn graph of detected genes in the ORF8 data set compared to published datasets (related to host antiviral genes / host cytokine genes; genes in APCs).
Conclusions
Overall, the results of the study demonstrate how the ORF8 protein interacts with DCs to cause a cytokine storm that led to ARDS in some severe cases of COVID-19. Therefore, prevention of ORF-8-mediated cytokine and chemokine response may lead to new treatment for severe COVID-19. Current treatments focus on single cytokines or SARS-CoV-2 neutralizing spike protein; however, the study suggests that neutralization of ORF8 protein may be a more promising approach to prevent progression to severe COVID-19.
Antibodies to ORF8 cannot neutralize the immunomodulatory function of ORF8 and even enhance binding to DCs, a phenomenon referred to as antibody-dependent enhancement or ADE. Studies have documented ADE during infection with several viruses, including influenza and human immunodeficiency virus-1 (HIV-1). In the context of SARS-CoV-2, numerous studies have demonstrated a high response to antibodies to the ORF8 protein. These antibodies can bind the fractional, crystallizing receptors (FcR) of DCs, leading to ADE. These findings may help to make ORF8 neutralizing or FcR blocking antibodies that could mitigate the effect of OFR8 on DC.
*Important message
bioRxiv publishes preliminary scientific reports that are not reviewed by partners and therefore should not be considered convincing, guiding clinical practice / health-related behavior, or treated as established information.
Reference journal:
- The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and causes a hyperinflammatory cytokine storm, Matthias Hamdorf, Thomas Imhoff, Ben Alan Bailey-Elkin, Janina Betz, Sebastian J. Theobald, Alexander Simonis, Veronica Di Cristasiano, Lutz Giselmann, Felix Dewald, Clara Lehmann, Max Augustine, Florian Klein, Miguel Alejandre Alcazar, Robert Rogisch, Mario Fabri, Jan Ribnicker, Heike Goebel, Kölgen Köfelvo, Jörg Goefelvo Koch, Felix Bock, bioRx 2022, DOI:
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