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The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and induces a hyper-inflammatory cytokine storm

The novel coronavirus pandemic, whose first outbreak was reported in December 2019 in Wuhan, China (COVID-19), is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Tissue damage caused by the virus leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In many viral infections the recruitment of monocytes into the lung and their differentiation to dendritic cells (DCs) are seen as a response to the viral infection. DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of the ORF8 protein, a specific SARS-CoV-2 open reading frame protein, with dendritic cells (DCs). We show that ORF8 binds to dendritic cells, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple pro-inflammatory cytokines by these cells. In addition, we identified dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as a possible interaction partner of ORF8 on dendritic cells. Blockade of ORF8 signaling leads to reduced production of IL-1β, IL-6, IL-12p70, TNF-α, MCP-1 (CCL2), and IL-10 by dendritic cells. Analysis of patient sera with high anti-ORF8 antibody titers showed that there was nearly no neutralization of the ORF8 protein and its function. Therefore, a neutralizing antibody that has the capacity of blocking the cytokine and chemokine response mediated by ORF8 protein might be an essential and novel additional step in the therapy of severe SARS-CoV-2 cases.


Introduction
The novel coronavirus pandemic, whose first outbreak was reported in December 2019 in Wuhan, China , is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1)(2)(3). This virus has risen to a global pandemic with over ~267.208 million infections and over ~ 5.272 million death (John Hopkins University, 8 th December 2021). The SARS-CoV-2 is a positive-stranded enveloped RNA virus that belongs to the family of Coronaviridae, a virus family with a natural reservoir in bats that has 55% nucleotide similarity and 30% protein sequence similarity with SARS-CoV, the virus that caused the first documented previous outbreak of severe acute respiratory syndrome in 2002 (4). During acute infection, the cytokine release syndrome (CRS) seems to be responsible for the severe conditions and the development of SARS (5). The disease is divided into two phases. During the non-severe stage, the virus triggers the innate and adaptive immune response. The innate immunity is mainly mediated by phagocytic cells, i.e. professional antigen-presenting cells, including dendritic cells (DCs), macrophages, and granulocytes that are residential in the lung or infiltrate into the lung tissue after infection (5). Especially the recruitment of monocytes into the lung and their differentiation into inflammatory, CD1c+, monocyte derived DCs (moDCs) plays a crucial role in viral infection like influenza (6) as well as SARS-CoV-2 (7,8). During this phase, the body begins to control the virus infection by an orchestrated T and B cell response and starts to eliminate the virus to suppress disease progression. However, if the body fails to develop a protective immune response during this early phase, the virus propagates, and massive destruction of the affected tissues occurs in the severe stage (9). The tissue damage caused by the virus leads to a strong response and activation of antigen-presenting cells. This second innate immune response can elicit acute respiratory distress syndrome (ARDS), which causes life-threatening respiratory disorders and is characterized by the rapid onset of widespread inflammation, the so-called cytokine storm (10). Dendritic cells are important players among other innate and adaptive immune cells in the development of the acute lung inflammation that causes ARDS (5,11,12) and a related cytokine storm. Therefore, these cells are center of this study.
Even if patients are not running into a severe acute respiratory syndrome with an uncontrolled inflammatory storm, SARS-CoV-2 infected patients suffer from a massive variety of long-term symptoms (13). The over-reactive immune system triggered by the infection leads to inflammation, and one particularly susceptible organ that is affected is the heart. One of the symptoms induced by inflammation is cardiomyopathy, in which the muscles of the heart become stretched, stiff, or thickened, affecting the heart's ability to pump blood. These symptoms are occurring in patients even after discharge from the hospital (14). The study of Carfi, A. et al. with 143 people that survive the infection with COVID-19 report as the most common lasting effect fatigue (53%) and shortness of breath (43%) (15). In 88% of the patients, visible damages of the lung are still detectable even six weeks after being discharged from the hospital, and even after 12 weeks, the number is only decreasing to 56%. The symptoms that are related to this long-lasting damage may take a longer time to fade and may still trigger a chronic inflammation (16,17). For the SARS-CoV-2 virus, the ongoing inflammation and the long-term clinical picture seems to differ from the two other known coronavirus infections in human, SARS-CoV and MERS-CoV (18). This leads to the question of how these viruses differ from each other.
The SARS-CoV-2 virus contains a unique new open reading frame 8 (orf8) gene that differs from the reading frames in SARS-CoV-1 and MERS-CoV (19). Several functions have been predicted for the ORF8 protein of SARS-CoV-2. In cell culture, ORF8 exogenous overexpression disrupts IFN-I signaling (20). It has been shown that ORF8 of SARS-CoV-2, but not ORF8 or ORF8a/b of SARS-CoV-1, is involved in the downregulation of MHC-I in cells (21).
A recent study focused on the secretion of the ORF8 protein from infected cells in vitro as well as secreted ORF8 protein in patients. Wang et al. showed that ORF8 is mostly a secretory protein. Additionally, the study showed that patients with a SARS-CoV-2 infection showed early seropositivity for anti-ORF8 IgM, IgG, and IgA and that ORF8 can be used as an early diagnostic marker (22). Since dendritic cells (DCs) and macrophages act as antigen-presenting cells (APCs), the infection of these cells by SARS-CoV-2 or the interaction with virus proteins may impair the adaptive immune responses against the virus and trigger the initial process of the cyto-/chemokine storm (23). The function and interaction of dendritic cells with virus proteins and their response are still poorly understood. Therefore, our study focus is on the interaction of the secreted ORF8 protein with dendritic cells and its contribution to the cytokine storm observed in COVID-19 patients (24).

Results
ORF8 specifically binds to monocytes and dendritic cells ORF8 contains a short signal peptide sequence similar to the one present in the S-protein.
Recently, it has been shown that ORF8 is secreted from infected cells, indicating the functionality of the signal peptide sequence (22). To confirm this result, we transfected HEK293 cells with the wild type ORF8 coding sequence, including the short 5' untranslated region. After induction for 24 h with doxycycline, ORF8 was detectable in the supernatant by coomassie blue staining ( Figure S2). Since CD14+ monocytes are able to recognize foreign proteins, we analyzed if the ORF8 protein can interact with this cell type. Therefore we used a recombinant ORF8 protein ( Figure S1 a, b) labeled with Atto488. We could show that within human peripheral blood mononuclear cells (PBMCs), mainly CD14+ monocytes bound ORF8 ( Figure 1B, n=3 and Figure S3 a). To exclude that this binding is a random protein binding we used labled BSA as a control. The binding of ORF8 to monocytes and related DCs is statistically much higher than to a random protein ( Figure   S3 a, b). As monocytes are the origin of the antigen-presenting dendritic cells (Figure 1 a), we followed up the binding of ORF8 to immature and mature dendritic cells. Both populations (n=3 donors for each experiment) have the ability to interact with the ORF8 protein, as shown in Figure 1 c and Figure S3 c. Monocytes, as well as dendritic cells, are known to bind and uptake antigens of a considerable variety of sources. To validate this specific binding of ORF8, we tested a commercially available rabbit anti-ORF8 polyclonal antibody for blocking. As shown, this polyclonal antibody led to reduced binding of ORF8 to immature and mature dendritic cells (Figure 1 d), confirming the specificity of this interaction. The corresponding isotype controle was not able to reduce ORF8 binding ORF8 overdrives DC differentiation into pre-maturity Dendritic cells are an important modulator of the immune response and may play an important role in the clearance of SARS-CoV-2 infections. As monocytes are recruited into the lung during infection and differentiate into inflammatory, monocyte derived DCs, we aimed to define the function of ORF8 as an immune modulator and virulence factor in the following part. In dendritic cell biology, antigens and especially superantigens from some viruses and bacteria can induce the differentiation of monocytes to dendritic cells (25,26) ( Figure 1 e). In our study, we found that ORF8 showed no influence on monocytes, similar to BSA, and is not acting as an inducer of differentiation ( Figure 1 f-k; n=3). Monocytic precursor cells treated with BSA or ORF8 remained positive for the monocyte marker CD14 (Figure 1 h, k respectively) and negative for the dendritic cell markers DC-SIGN ( Figure 1 f, i, respectively), and CD1c (Figure 1 g, j respectively).
Next, we analyzed if ORF8 can influence the differentiation process. Therefore, we differentiated monocytes to immature dendritic cells (immature MoDC) with GM-CSF and IL-4 in the presence or absence of ORF8 in different concentrations (Figure 2 a). We performed a dose escalation of ORF8 protein and looked for the upregulation of the maturation marker CD40 and CD80. A first plato phase was reached between 500 and 1000 ng/ml and had an additional small increase at 2000 ng/ml ( Figure S4 c). Based on that we chose a concentration of 1000 ng/ml for all further experiments to elict a robust activation. ORF8 treated cells showed a significantly different stellate-like morphology which is comparable to mature dendritic cells ( Figure S8 a, b). In line with the morphological changes, also the expression of the maturation markers MHCII, CD83, CD80, CD86, and CD40 were markedly upregulated. In contrast, the dendritic marker CD11c was slightly downregulated (Figure 2 b, total n=6). The surface markers of a conventional induced maturation are shown in Figure S8 c. Neither BSA, nor the polyclonal anti-ORF8 antibody alone, its isotype control or a human Fc-antibody fragment as controls were able to induce maturation (Figure S3 e).
In addition, we also analyzed the DC specific marker DC-SIGN in this experimental setup.
Interestingly, the expression of this marker was almost completely downregulated ( Figure   2 c; n=3).
To verify the specific effect of ORF8 on the expression of DC-SIGN we used a polyclonal anti-ORF8 antibody. Neutralizing ORF8 with this polyclonal antibody partially restored the expression of DC-SIGN. These results demonstrate that the effect is regulated by the ORF8 induces a unique pro-inflammatory cytokine secretion during DC differentiation SARS-CoV-2 can induce a cytokine storm leading to ARDS (10). Therefore, we also analyzed the chemokine and cytokine expression profile of dendritic cells exposed to ORF8 during differentiation. Strikingly, we found a significant, strongly elevated secretion of IP-10, IL-1β, TNF-α, MCP-1, IL-6, IL-10, and IL12p70 (Figure 3 a) as well as an increased level of IFN- and IL-8 (Fig. S12 b) in comparison to untreated cells (0 µg/ml ORF8). This effect could be partly reversed by the simultaneous addition of a neutralizing polyclonal rabbit antibody against ORF8 during the differentiation, verifying the ORF8 specific induction of the cytokine storm ( Figure S6). Parallel to the exclusion of contamination of the purified ORF8 protein with endotoxin ( Figure S1  Using the metascape platform for enrichment and pathway analysis (28), we could show that this cytokine profile is significantly associated with COVID-19 adverse outcome pathways as well as SARS-CoV-2 innate immunity evasion and cell-specific immune response pathways (Figure 3

b).
To get deeper insights into the function and underlying mechanism of ORF8, we performed RNA sequencing of DCs that were differentiated in the presence or absence of ORF8 (120 h). We found a total of 843 regulated genes (p-value <0.05 after Benjamimi Hochberg correction) when comparing immature DCs with DCs, which received ORF8 during the differentiation (Figure 4 a, each group n = 4). Subsequent pathway and process enrichment analysis (metascape platform) of the ORF 8 activated DC RNA profile revealed a strong overlap with inflammatory and immune-activating pathways (Figure 4 b, S7 a). Next, we compared the regulated genes in ORF 8 treated DCs with regulated antiviral-related genes and cytokine genes in host cells after SARS-CoV2 infection (29).

Lack of ORF8 neutralizing antibodies in COVID-19 patients
As demonstrated, ORF8 showed a strong immunomodulatory function in our study, and we were interested if patients infected with the SARS-CoV-2 virus produce antibodies against ORF8. Therefore we performed a custom-made ELISA to detect ORF8 antibodies in the convalescent plasma of 64 COVID-19 patients early tested positive for SARS-CoV-2 by RT-PCR (0-90 days). We found eight patients highly positive for anti-ORF8 IgG antibodies ( Figure 5 a). To confirm this finding, another set of plasma (n=104) was analysed for the content of anti-ORF-8 antibodies. Again, eight patients were highly positive for anti-ORF8 IgG antibodies (Figure 5 c). Here, seven out of the eight samples also had a very strong reaction toward the SARS-CoV-2 spike protein ( Figure S12). Next, we compared the serum samples of the ORF8 high and low/negative patients regarding the expression of pro-inflammatory chemokines and cytokines. In line with our in vitro findings, we found a significantly increased expression of IP-10 ( Figure 5B) (p-value < 0.05 after). An overview of all performed antibody, PCR and the neutralization assays on patient serum can be found in the supplementary table (Table S1) and in the Spike (RBD) ELISA (Fig S12 a).
As we demonstrate, ORF8 shows a robust immunomodulatory capacity, and patients produce antibodies against ORF8; we analyzed if these antibodies from SARS-CoV-2 infected patients have the potential to neutralize the ORF8 effect on dendritic cells.
Therefore, eight sera with high ORF8 antibody titer from the patient collective of late time points ( Figure 5 c, labeled in blue) were analyzed. We pre-incubated the ORF8 protein with 5 µl of the serum samples (with correspond to an estimated anti-ORF8 antibody concentration between 1 -5 µg) of each donor overnight to ensure the binding of the anti-ORF8 antibodies. The monocytes were incubated with the ORF8/sera complex or only serum during the differentiation. As a positive control, ORF8 without sera was used.
We analyzed the ability of the sera to inhibit the binding and cyto-/chemokine production induced by ORF8. We were able to show that the antibodies directed against ORF8 could not reduce the binding of ORF8 to DCs (Figure 6 a). Only one of the donors (green box) had the capacity to neutralize this effect (Figure 6 a) and showed no enhanced binding of ORF8 to DCs (Serum 396, Figure 6 a). We analyzed this effect in more detail and seven out of the eight sera could not reduce the entire range in cyto-/chemokine production that is triggered by ORF8 (Figure 6 b). Analogous to our previous observation (Figure 2 b), we analyzed the surface expression of dendritic cell-specific markers and activation markers.
We could show that the anti-ORF antibody-positive patient sera were not able to reduce ORF8 induced DC activation (Figure 6 c). Instead, we observed for most of the sera an enhancement of the ORF8 induced upregulation of MHC II, CD80, and CD83. By preincubating ORF8-Atto488 with anti-ORF8 IgG + sera, we could show that ORF8 bound even better in combination with this sera. This effect could be reversed with blockade of the Fc receptor on the DCs ( Figure S9).
In addition, in a small case series with sera from three patients hospitalized due to the severity of their SARS-CoV-2 infection, we found that, with the progression of the infection, the ORF8 induced cytokine storm even aggravated in the presence of anti-ORF8 IgG + sera over time ( Figure S10).

Plasticity of the antibody recognized loop region of ORF8
The advantage of our study is that we used for the first time ORF8 expressed and purified from a mammalian system, whereas previous studies used bacterial-derived proteins (33).
To compare the proteins and possible structural differences and a different potential structure for binding antibodies, we crystallized the eukaryotic expressed ORF8 protein and compared the structure against the bacterial expressed SARS-CoV-2 ORF8 protein and bat-related ORF8 variants.
The crystal structure of a SARS-CoV-2 ORF8 Asn78Gln mutant (ORF8 N78Q ) purified from HEK293 cells was determined to 2.6Å. The overall structure of ORF8 N78Q is comparable to the previously reported ORF8 (33) structure produced in E. coli, with some minor differences. The asymmetric unit is comprised of a single ORF8 monomer, with a second monomer related by a crystallographic 2-fold axis generating the biological dimer, linked by a disulfide bond at the interface (Figure 7 a). Each ORF8 monomer adopts an Ig-like fold comprising a pair of anti-parallel β-sheets held together by three disulfide bonds.
Interestingly, while the ORF8 dimer interface is essentially identical to the previously reported structure, our ORF8 N78Q structure shows a difference in the relative orientation of the two monomers within the covalent dimer, with a rotation of ~20° seen between monomers (Figure 7 b), suggesting that the ORF8 dimer interface is somewhat dynamic, despite the extensive, disulfide-bonded interface. This rotation gives the ORF8 N78Q structure a more "closed" shape, bringing the surface loops and surrounding structure between β2-β3 and β7-β8 of either monomer closer, towards the dimer interface (Figure   7 b). In addition, several solvent-exposed loop structures between strands β2-β3, β3-β4, β4-β5, and β7-β8 (numbered according to Flower et al. 2021) adopt slightly different conformations compared to deposited structures (Figure 7 c). In particular, residues 63-77, which includes the SARS-CoV-2-specific 73YIDI76 motif postulated to mediate ORF8 oligomerization (33), were unable to be modelled due to lack of electron density in the region (Figure 7 a). This is consistent with a previous report describing significant structural plasticity in this loop structure (33).
To further understand the structure of this loop region and possible consequences for the accessibility of antibodies, homology models were built for the ORF8 homologues of

Discussion
Recently, it was shown that ORF8 is secreted by SARS-CoV-2 infected cells and could be detected in the serum of COVID-19 patients (22). Furthermore, it was recently reported that a mutant SARS-CoV-2 strain found in Singapore displayed a deletion of 382 nucleotides in the region of ORF8 associated with decreased pathogenicity (34) (24) among several point mutations and smaller deletions (35)(36)(37), indicating the importance of ORF8 in the pathogenicity of this infectious disease. All main strands of SARS-CoV-2 contain the ORF8 protein (https://covariants.org/).
To study the patho-mechanistic role of ORF8 in COVID-19, we first investigated whether ORF8 is strongly expressed in infected tissue and is detectable in sera ( Figure S4 b, a) as shown in the literature by Xiaosheng et al (38) and if ORF8 can directly bind to blood cells. PBMCs are composed of lymphocytes and monocytes, precursor cells for macrophages, and dendritic cells. In this study, we were able to demonstrate that extracellular ORF8 protein binds specifically to DCs (immature and mature, Figure 1 c, d, As ORF8 directly binds to monocytes, we were interested if ORF8 can also directly trigger the differentiation of monocyte to DCs. For several viruses, e. g. HIV-1 (39), it is known that their proteins are able to modulate dendritic cells and their function. Our findings show that ORF8 alone is not able to trigger differentiation since the precursor cells remained in a CD14 + monocyte stage (Figure 1 k). Since ORF8 independently was not able to modulate monocytes, we analyzed if ORF8 has an impact on the differentiation from monocytes to DCs. In fact, we could show that precursor monocytes differentiated by IL-4 and GM-CSF in the presence of the full-length ORF8 protein induced a pre-maturity in the differentiated DCs characterized by a mature cell morphology (40) (43). Even more interesting, it was also shown to be key for SARS-CoV-2 infection of DCs (44). From HIV infection, DC-SIGN is known to form an infectious synapse between infected DC and T cells that facilitates HIV infection (45). Furthermore, it was shown that DC-SIGN mediates the internalization of intact HIV into a low pH nonlysosomal compartment in dendritic cells. This internalization is needed for the trans-activation of T cells by DCs (27).
In this study, we could show that blockade of ORF8 partly rescues the expression of DC-SIGN on the surface of immature DCs (Figure 2 d), pointing towards the interaction of ORF8 and DC-SIGN as described for HIV and DC-SIGN. In fact, we could further show that ORF8 interacts directly with DC-SIGN in a co-immune precipitation assay and that this interaction is reduced by an anti-ORF8 neutralizing antibody (Figure 2 e). Hence, DC-SIGN is a potential receptor for ORF8 and might be internalized upon binding to ORF8. DC-SIGN is known for its immune regulative role in DCs (46) and macrophages (47).  (59). Nonetheless, in accordance with our study, the cohort was divided into patients with high and low levels of ORF8 antibodies. Since the results of the study are waiting for validation by a conventional ELISA, further studies might explain the different findings.
Nevertheless, we could nearly not detect a neutralizing capacity of the identified anti-ORF8 IgG + sera on the ORF8 induced cytokine storm. Instead, we found an enhanced binding of ORF8 pre-incubated with anti-ORF8 IgG + sera to DCs (Figure 6 a). This enhancement seems to be mediated by the Fc-receptors on the dendritic cells since it could be blocked by conventional Fc-block ( Figure S9). So, the production of anti-ORF8 antibodies seems to be contra-productive to inhibit the ORF8 mediated effect. Yajing Fu et al. discussed various mechanisms of SARS-CoV-mediated inflammation potential therapeutic tools to reduce SARS-CoV-2-induced inflammatory responses, including multiple methods to block FcR activation (60). In our study, we demonstrated that in most patients, antibodies against ORF8 cannot neutralize the immune-modulatory function of ORF8 (Figure 6 b, c) and even enhanced the binding (antibody-dependent enhancement (ADE)) of the protein to DCs (Figure 6 a, b, and c). For patients with severe SARS-CoV-2 infection in the intensive care unit, we even found that the effect gets enhanced with the progression of the disease ( Figure S10). This effect is documented for several viruses like influenza (61), Dengue (62) as well as HIV-1 (63) (virus-induced ADE). ADE is intensively discussed for SARS-CoV-2 infection, and many studies demonstrated that antibodies against the virus were key to enhance the infection rate and are a critical challenge for developing further vaccines (64)(65)(66). In our study, we could show that the ORF8 protein induces in several patients a high antibody response ( Figure 5) against the ORF8 protein and that these antibodies can bind to the FcR receptors ( Figure S9) of dendritic cells and leads to an antibody-dependent enhancement (Figure 6 a). Therefore, an ORF8 neutralizing antibody or the use of FcR blocking antibodies could be beneficial for the outcome of severe SARS-CoV-2 infections. So, the detection of ORF8 antibodies in patient serum underscores the importance of our finding that ORF8 is an important factor in the development of COVID-19 and the missing neutralizing capacity of the serum antibodies shows that there is an unmet need to develop a neutralizing anti-ORF8 antibody to block , amongst others, the proinflammatory effect of ORF8 on dendritic cells.
Since we used, for the first time, SARS-CoV-2 ORF8 purified from HEK293 cells that include all protein modifications like e.g. glycosylation, we determined the structure of this protein to a resolution of 2.6Å and observed that the overall structure of ORF8 is consistent with published structures of ORF8 isolated from E. coli (33). Interestingly, we noted a considerable degree of flexibility between monomers within the ORF8 dimer We show that ORF8 causes the pre-maturation and activation of differentiating DCs and induces the secretion of pro-inflammatory cytokines. We further looked into the neutralizing antibody capacity of patient sera to neutralize the ORF8 induced effect.
In conclusion, our findings show that ORF8 contributes to the cytokine and chemokine storm described for patients with SARS-CoV-2 infections (54, 67-69) by interaction with dendritic cells. Furthermore, our findings shed light on the function of ORF8 and its role in the development of ARDS.
Blocking of the cytokine and chemokine response mediated by the ORF8 protein might be an essential and novel additional step in the therapy of severe SARS-CoV-2 cases.
Our findings showed that a polyclonal rabbit antibody against ORF8 is able to strongly reduce the binding of ORF8 (Figure 1 d) and the humoral immune response of dendritic cells ( Figure S6). This demonstrates that the observed effects are a direct response to the ORF8 protein. Current treatment options focus on neutralizing single cytokines, chemokines (69), or the spike protein, but we could show for the first time evidence that neutralizing ORF8 and its strong pro-inflammatory characteristics is a promising approach to take out one of the possible key players that cause the cytokine storm. Kreer et al. (70) recently reported the longitudinal isolation of potent S protein neutralizing antibodies from COVID-19 patients. This strategy might also be promising in identifying ORF8 neutralizing antibodies. As antibodies failed to prevent or even enhance ORF8 binding to DCs ( Figure   6), an experimental structure of ORF8 isolated under non-denaturing conditions confirms the stoichiometry of ORF8 and the biological relevance of the isolated disulfide-linked homodimer relevant structure is important for the further development of inhibitory antibodies targeting ORF8. Unfortunately, it is difficult to ascertain how the flexibility of ORF8 would influence antibody binding in the absence of an experimental structure of ORF8 bound to an antibody of interest. The apparent flexibility of this region suggests that it may adopt alternate conformations upon binding to an interaction partner. As suggested elsewhere (33), this region of ORF8 may also contribute directly to the formation of higherorder oligomers, although the relevance of such structures has not been demonstrated directly. In the event that such structures are functionally important for SARS-CoV-2 pathogenesis, antibody-mediated disruption of these complexes may be important.
In general, we conclude that ORF8 via binding to dendritic cells is an important player in SARS-CoV-2 induced chemokine and cytokine storm in patients with severe outcomes and that targeting ORF8 may open new treatment avenues for severe COVID-19 patients (graphical abstract Figure S12).

Protein design, expression, and purification
The ORF8 open reading frame was cloned into a sleeping beauty transposon vector either with or without a Strep-tag at the N-term (Figure S1  GraphPad Software, San Diego, USA) was used and a one-and tow-way ANOVA statistic was performed.

Polyclonal antibody against ORF8
Recombinant ORF8 was used to immunize two rabbits. After two months, sera were collected and the antibodies affinity purified via an ORF8 affinity column. Tag free ORF8 was coupled to CNBr-activated Sepharose (GE Health-care), and after applying the sera to the column the specific antibodies were eluted with 150 mM NaCl, 0.1 M triethylamine, pH 11.5 and neutralized with 1M Tris-HCl, pH 6.8. The two antibodies were tested by Western blot analysis. 25 µl of the HEK293 supernatants were applied to a 12% SDS-PAGE gel under reducing conditions and visualized either by Coomassie Blue staining or by Western blot analysis utilizing the ORF8 affinity-purified antibodies from rabbit 1.

Isolation and differentiation of monocytes to dendritic cells.
Mononuclear cells (PBMCs) were freshly isolated using a density gradient (Lymphoprep, the density of 1.077 g/ml, Stemcell, France) from leukocyte concentrate (leukapheresis apheresis chamber). Ethical clearance for the use of human material was obtained from the ethical review committee of the university hospital Cologne (N°11-339). The harvested PBMCs were washed with PBS and resuspended in X-Vivo 15 (Biozym, Germany). The viability and cell number were obtained by trypan blue staining and counting. For the enrichment of monocytes, we used the adherence strategy as described before (71). For each well of a 6-well plate, we seeded 2 x 10 7 PBMCs in 2 ml of X-Vivo media. The cells were incubated for 75 min at 5% CO 2 and 90% humidity at 37˚C to allow adherence of monocytes. The non-adherent fraction (NAF) was removed from the 6-well plates by intensive washing. For differentiation to immature moDCs, X-Vivo 15 media was supplemented with 100 ng/ml human granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng/ml human interleukin 4 (IL-4) (both cytokines obtained from Peprotech, Germany), and cells were cultured with 5% CO 2 , and 90% humidity at 37˚C

Blocking of ORF8 by polyclonal rabbit Ab
For blocking the labeled and unlabelled ORF8 protein, we pre-incubated the protein with an anti-SARS-CoV-2 ORF8 detecting antibody (Antibody.com, London, UK) for half an hour on ice to induce ORF8/antibody interaction. Due to the sodium azide in the antibody solution, we used the same µg of the antibody (max. 2 µg) as µg of the ORF8 protein. In addition, we used the antibody alone in the same concentration as in the complex as an additional control for cytotoxicity. As an isotype control an unspecific polyclonal rabbit antibody was used (NEB, Frankfurt, Germany). To block ORF8 during differentiation it was preincubated with the polyclonal rabbit anti-ORF8 antibody or isotype over night and added to the monocytes (t=0) at the same time as GM-CSF and IL-4 for the differentiaton.

Co-immunoprecipitation of ORF8 and DC-SIGN
For the co-IPs 25 µl of streptavidin-coated magnetic beads (NEB, Frankfurt, Germany) were used per separation. The beads were washed with 500 µl PBS once before use and resuspended in 50 µl PBS. For immune precipitation (IP), beads were pre-incubated at 4°C for 1h with agitation with empty control (no protein) or 2 µg ORF8 protein with Strep II tag or 2 µg ORF8 plus 2 µg of an anti-ORF8 antibody (Antibody.com, London, UK).
Unbound protein was washed from the beads with 500 µl PBS, spun down and resuspended in 50 µl PBS. For protein-protein interaction, pre-incubated beads were incubated a second time with 2 µg of a recombinant human DC-SIGN protein with a human Fc tag (Sino Biological, Köln, Germany) for 1 h at 4°C with agitation. The beads were washed three times with 500 µl PBS before denaturation and separation by a polyacrylamide gel (4-12% gradient). The gels were blotted to a nitrocellulose membrane and blocked with 5 % milk in TBST buffer. For the staining, a direct HRP conjugated antihuman Fc (Invitorgen, Wiesbaden, Germany) was used, and bands were detected with an imager (Bio-Rad, Feldenkirchen, Germany).

Immuno blot and immuo histochemistry
Circulation of the ORF8 was analyzed in sera of five hospitalized patients collected at two timepoints (t1 and t2). Therefore, 2 µl of sera was boild in 22 µl dendaturing laemmli loading buffer and seperated with a 4 -12% NuPage SDS gel, running with MES buffer.
As a control healthy donor sera was spiked in with bacteria expressed ORF8 (RP-87666; Invitrogen, Darmstadt, Germany) with the indicated amount/ml. The seperated protein was transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk for one hour, followed by the primary antibody 1:1000 (anti-ORF8 C-term (ABIN6992307), antibodies-online.com, Aachen, Germany) over night. As a secondary antibody we used an F(ab´) 2 anti-rabbit HRP 1:7000 (NA9340, Amersham, Freiburg, Germany). The development was performed with West Pico PLUS Chemiluminescent Substrate (Thermo, Darmstadt, Germany) with an imaging system from bio-rad (Bio-rad, Feldkirchen, Germany). (w/v) polyethylene glycol (PEG) 3350. A single crystal was swept through a dropcontaining well solution supplemented with 20% glycerol and mounted directly into the cryostream. X-ray diffraction images were collected using a Rigaku 007-HF MicroFocus X-ray generator and an R-AXIS IV++ image plate detector. X-ray diffraction images were integrated and scaled using XDS (73), and merged using Aimless (74) within the CCP4i2 software package (75). Initial phase estimates for the ORF8 N78Q structure were obtained by molecular replacement within Phaser (76), using a modified polyalanine search model derived from PDB entry 7JTL (33). An initial model was build using AutoBuild (77), and successive rounds of model building and refinement were performed using Coot (78) and phenix.refine (79), respectively. Coordinates and structure factors for the ORF8 N78Q have been deposited to the Protein Data Bank under PDB ID 7MX9, and X-ray data reduction and refinement statistics are listed in Table 1. Structural models for additional CoV ORF8 homologues were built using Modeller (80) with sequences acquired from NCBI accession numbers AGZ48826.1 (bat SARS-like CoV Rs3367), AHX37565.1 (Bat-CoV RaTG13) and AAU04641.1 (SARS-CoV). All structures were modeled using chain A of the previously reported and deposited ORF8 structure 7JTL (33) as a template. The Illumina® TruSeq® mRNA stranded sample preparation Kit was used for library preparation. Briefly, mRNA was purified and fragmented from 1µg total RNA after poly-A selection. After reverse transcription using random primers, second strand cDNA synthesis was performed and, after end repair and A-tailing, indexing adapters were ligated. Purified and amplified products were used to create the final cDNA libraries.

Acknowledgments
We thank Semra Oezcelik for her excellent technical assistance. We owe special thanks

SARS-CoV-2 outcomes. We hope that our study will help to ensure that their story
is not forgotten and that it will be possible to prevent severe cases in the future. We              datasets (host antiviral-related genes/host cytokine genes (29); genes in APCs (30)).

Figure S9: Enhanced binding of ORF8 is antibody-mediated via Fc receptors
The binding capacity of ORF8-Atto488 or ORF8-Atto488 in the presence of sera from anti-ORF8 positive or healthy donors was determined on immature dendritic cells by FACS analysis. The effect of the Fc receptors was determined by blocking them with a commercially available Fc block