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The recent development of solvent- and polymer-based brain clearing techniques has greatly advanced our ability to visualize the mammalian nervous system in 3D. However, it remains challenging to image the mammalian body en bloc as existing methods can be prohibitively expensive or difficult in practice. Here, we developed HYBRiD (HYdrogel-Based Reinforcement of DISCO), by strategically recombining components of organic- and polymer-based clearing pipelines and achieved superb simplicity, transparency, protein retention, and compatibility with direct fluorescent imaging as well as immunostaining in cleared mammalian bodies. Using parvalbumin- and somatostatin-Cre models, we demonstrated the utility of HYBRiD for whole-body imaging of genetically encoded fluorescent reporters without antibody enhancement of signals in newborn and juvenile mice. Using K18-hACE2 transgenic mice, HYBRiD enabled perfusion-free clearing and visualization of SARS-CoV-2 infection in a whole mouse chest, revealing macroscopic and microscopic features of viral pathology in the same sample. HYBRiD offers a simple, affordable, and universal solution to visualize large heterogeneous body parts or entire animals for basic and translational research.

SARS-CoV-2 is the coronavirus pathogen of the currently prevailing COVID-19 pandemic. It relies on its main protease (MPro) for replication and pathogenesis. MProis a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as MProinhibitors. However, dipeptidyl inhibitors especially those with a spiro residue at their P2 position have not been systematically investigated. In this work, we synthesized about 30 reversibly covalent dipeptidyl MProinhibitors and characterized them onin vitroenzymatic inhibition potency, structures of their complexes with MPro, cellular MProinhibition potency, antiviral potency, cytotoxicity, andin vitrometabolic stability. Our results indicated that MProhas a flexible S2 pocket that accommodates dipeptidyl inhibitors with a large P2 residue and revealed that dipeptidyl inhibitors with a large P2 spiro residue such as (S)-2-azaspiro[4,4]nonane-3-carboxylate and (S)-2-azaspiro[4,5]decane-3-carboxylate have optimal characteristics. One compound MPI60 containing a P2 (S)-2-azaspiro[4,4]nonane-3-carboxylate displayed high antiviral potency, low cellular cytotoxicity, and highin vitrometabolic stability and can be potentially advanced to further preclinical tests.

SARS-CoV-2 virus has recently given rise to the current COVID-19 pandemic where infected individuals can range from being asymptomatic, yet highly contagious, to dying from acute respiratory distress syndrome. Although the world has mobilized to create antiviral vaccines and therapeutics to combat the scourge, their long-term efficacy remains in question especially with the emergence of new variants. In this work, we exploit a class of compounds that has previously shown success against various viruses. A salicylanilide library was first screened in a SARS-CoV-2 activity assay in Vero cells. The most efficacious derivative was further evaluated in a prophylactic mouse model of SARS-CoV-2 infection unveiling a salicylanilide that can reduce viral loads, modulate key cytokines, and mitigate severe weight loss involved in COVID-19 infections. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and a previously established favorable pharmacokinetic profile for the lead salicylanilide renders salicylanilides in general as promising therapeutics for COVID-19.

Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and, thus, the spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model and, thus, provide a novel avenue to pursue therapy.

The exploration and identification of safe and effective vaccines for the SARS-CoV-2 pandemic have captured the world's attention and remains an ongoing issue due to concerns of balancing protection against emerging variants of concern while also generating long-lasting immunity. Here, we report the synthesis of a novel messenger ribonucleic acid encoding the spike protein in a lipid nanoparticle formulation (STI-7264) that generates robust humoral and cellular immunity following immunization of C57Bl6 mice. In an effort to improve immunity, a clinically focused lymphatic drug delivery device (MuVaxx) was engineered to modulate immune cells at the injection site (epidermis and dermis) and draining lymph node (LN) and tested to measure adaptive immunity. Using MuVaxx, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine-producing CD8 T cells, neutralizing antibodies against the Washington (wild type) strain and South African (Beta) variants, and LN-resident spike-specific memory B cells. Remarkably, a 4-fold-elevated T cell response was observed in MuVaxx-administered vaccination compared to that of IM-administered vaccination. Thus, these data support further investigation into STI-7264 and lymphatic-mediated delivery using MuVaxx for SARS-CoV-2 and VoC vaccines.

BACKGROUND/AIMS: During an immune response, type I interferon (IFN-I) signaling induces a wide range of changes, including those which are required to overcome viral infection and those which suppress cytotoxic T cells to avoid immunopathology. During certain bacterial infections, IFN-I signaling exerts largely detrimental effects. Although the IFN-I family of proteins all share one common receptor, biologic responses to signaling vary depending on IFN-I subtype. Here, we asked if one IFN-I subtype dominates the pro-bacterial effect of IFN-I signaling and found that control of Listeria monocytogenes (L.m.) infection is more strongly suppressed by IFN-β than IFN-α. METHODS: To study this, we measured bacterial titers in IFNAR-/-, IFN-β‑/‑, Stat2-/-, Usp18fl/fl and Usp18fl/fl x CD11c-Cre mice models in addition to IFN-I blocking antibodies. Moreover, we measured interferon stimulated genes in bone marrow derived dendritic cells after treatment with IFN-α4 and IFN-β. RESULTS: Specifically, we show that genetic deletion of IFN-β or antibody-mediated IFN-β neutralization was sufficient to reduce bacterial titers to levels similar to those observed in mice that completely lack IFN-I signaling (IFNAR-/- mice). However, IFN-α blockade failed to significantly reduce L.m. titers, suggesting that IFN-β is the dominant IFN-I subtype responsible for the pro-bacterial effect of IFN-I. Mechanistically, when focusing on IFN-I signals to dendritic cells, we found that IFN-β induces ISGs more robustly than IFN-α, including USP18, the protein we previously identified as driving the pro-bacterial effects of IFN-I. Further, we found that this induction was STAT1/STAT2 heterodimer- or STAT2/STAT2 homodimer-dependent, as STAT2-deficient mice were more resistant to L.m. infection. CONCLUSION: In conclusion, IFN-Β is the principal member of the IFN-I family responsible for driving the pro-bacterial effect of IFN-I.

As an essential enzyme of SARS-CoV-2, the COVID-19 pathogen, main protease (M

Boceprevir is an HCV NSP3 inhibitor that was explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (M

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant and its BA.X lineages, has rendered ineffective a number of previously FDA emergency use authorized SARS-CoV-2 neutralizing antibody therapies. Furthermore, those approved antibodies with neutralizing activity against Omicron BA.1 are reportedly ineffective against the subset of Omicron subvariants that contain a R346K substitution, BA.1.1, and the more recently emergent BA.2, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern.Following a campaign of antibody discovery based on the vaccination of Harbor H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167.STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against each of the tested Omicron subvariants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice.With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for intravenous or intranasal use in human clinical trials.Funded by CRIPT (no. 75N93021R00014), DARPA (HR0011-19-2-0020), and NCI Seronet (U54CA260560).

The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the efficacy and effectiveness of the two FDA-approved mRNA vaccines. Here, we explored various strategies to develop novel mRNAs vaccines to achieve safer and wider coverage of VOCs. Firstly, we constructed a cohort of mRNAs that feature a furin cleavage mutation in the spike (S) protein of predominant VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2). Not present in the mRNA vaccines currently in use, the mutation abolished the cleavage between the S1 and S2 subunits, potentially enhancing the safety profile of the immunogen. Secondly, we systematically evaluated the induction of neutralizing antibodies (nAb) in vaccinated mice, and discovered that individual VOC mRNAs elicited strong neutralizing activity in a VOC-specific manner. Thirdly, the IgG produced in mice immunized with Beta-Furin and Washington (WA)-Furin mRNAs showed potent cross-reactivity with other VOCs, which was further corroborated by challenging vaccinated mice with the live virus of VOCs. However, neither WA-Furin nor Beta-Furin mRNA elicited strong neutralizing activity against the Omicron variant. Hence, we further developed an Omicron-specific mRNA vaccine that restored protection against the original and the sublineages of Omicron variant. Finally, to broaden the protection spectrum of the new Omicron mRNA vaccine, we tested the concept of bivalent immunogen. Instead of just fusing two RBDs head-to-tail, we for the first time constructed an mRNA-based chimeric immunogen by introducing the RBD of Delta variant into the entire S antigen of Omicron. The resultant chimeric mRNA was capable of inducing potent and broadly acting nAb against Omicron (both BA.1 and BA.2) and Delta, which paves the way to develop new vaccine candidate to target emerging variants in the future.

The continual emergence of SARS-CoV-2 variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant, has rendered ineffective a number of previously EUA approved SARS-CoV-2 neutralizing antibody therapies. Furthermore, even those approved antibodies with neutralizing activity against Omicron are reportedly ineffective against the subset of Omicron variants that contain a R346K substitution, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. Following a campaign of antibody discovery based on the vaccination of Harbour H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of Spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against the Omicron and Omicron + R346K variants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for use in human clinical trials.

The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we use a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduced the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for anti-viral indications could benefit from in vitro affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.

As an essential enzyme to SARS-CoV-2, main protease (M (Pro) ) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 sites and the N -terminal protection group, we synthesized a series of M (Pro) inhibitors that contain β -(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 site. These inhibitors have a large variation of determined IC (50) values that range from 4.8 to 650 nM. The determined IC (50) values reveal that relatively small side chains at both P2 and P3 sites are favorable for achieving high in vitro M (Pro) inhibition potency, the P3 site is tolerable toward unnatural amino acids with two alkyl substituents on the α -carbon, and the inhibition potency is sensitive toward the N -terminal protection group. X-ray crystal structures of M (Pro) bound with 16 inhibitors were determined. All structures show similar binding patterns of inhibitors at the M (Pro) active site. A covalent interaction between the active site cysteine and a bound inhibitor was observed in all structures. In M (Pro) , large structural variations were observed on residues N142 and Q189. All inhibitors were also characterized on their inhibition of M (Pro) in 293T cells, which revealed their in cellulo potency that is drastically different from their in vitro enzyme inhibition potency. Inhibitors that showed high in cellulo potency all contain O - tert -butyl-threonine at the P3 site. Based on the current and a previous study, we conclude that O - tert -butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for peptidyl aldehyde inhibitors of M (Pro) . This finding will be critical to the development of novel antivirals to address the current global emergency of concerning the COVID-19 pandemic.

Boceprevir is an HCV NSP3 inhibitor that has been explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (M (Pro) ) and contains an α-ketoamide warhead, a P1 β-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert -butyl-glycine, and a P4 N -terminal tert -butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based M (Pro) inhibitors including PF-07321332 and characterized their M (Pro) inhibition potency in test tubes ( in vitro ) and human host cells ( in cellulo ). Crystal structures of M (Pro) bound with 10 inhibitors and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a β-(S-2-oxopyrrolidin-3-yl)-alanyl (opal) residue and the warhead with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N -terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the M (Pro) active site cysteine. The P1 opal residue, P2 dimethylcyclopropylproline and P4 N -terminal tert -butylcarbamide make strong hydrophobic interactions with M (Pro) , explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains an P4 N -terminal isovaleramide. In its M (Pro) complex structure, the P4 N -terminal isovaleramide is tucked deep in a small pocket of M (Pro) that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency in inhibiting ectopically expressed M (Pro) in human 293T cells. All inhibitors including PF-07321332 with a P4 N -terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N -terminal cap is changed to a carbamate. The installation of a P3 O-tert -butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N -terminal carbamate were advanced to antiviral tests on three SARS-CoV-2 variants. They all have high potency with EC (50) values around 1 μM. A control compound with a nitrile warhead and a P4 N -terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N -terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.

As an essential enzyme to SARS-CoV-2, main protease (MPro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 sites and the N-terminal protection group, we synthesized a series of MPro inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 site. These inhibitors have a large variation of determined IC50 values that range from 4.8 to 650 nM. The determined IC50 values reveal that relatively small side chains at both P2 and P3 sites are favorable for achieving high in vitro MPro inhibition potency, the P3 site is tolerable toward unnatural amino acids with two alkyl substituents on the α-carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of MPro bound with 16 inhibitors were determined. All structures show similar binding patterns of inhibitors at the MPro active site. A covalent interaction between the active site cysteine and a bound inhibitor was observed in all structures. In MPro, large structural variations were observed on residues N142 and Q189. All inhibitors were also characterized on their inhibition of MPro in 293T cells, which revealed their in cellulo potency that is drastically different from their in vitro enzyme inhibition potency. Inhibitors that showed high in cellulo potency all contain O-tert-butyl-threonine at the P3 site. Based on the current and a previous study, we conclude that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for peptidyl aldehyde inhibitors of MPro. This finding will be critical to the development of novel antivirals to address the current global emergency of concerning the COVID-19 pandemic.

The exploration and identification of safe and effective vaccines for the SARS-CoV-2 pandemic has captured the world’s attention and remains an ongoing issue in order to protect against emerging variants of concern (VoCs) while generating long lasting immunity. Here, we report the synthesis of a novel messenger ribonucleic acid (mRNA) encoding the spike protein in a lipid nanoparticle formulation (LNP) (STI-7264) that generates robust humoral and cellular immunity following immunization of C57Bl6 mice. In efforts to continually improve immunity, a lymphatic drug delivery device (MuVaxx) was engineered and tested to modulate immune cells at the injection site (epidermis and dermis) and draining lymph node (LN) to elicit adaptive immunity. Using MuVaxx, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine producing CD8 T cells, and neutralizing antibodies against the Washington (Wild Type, WT) and South African (beta) variants. Remarkably, a 4-fold elevated T cell response was observed in MuVaxx administered vaccination as compared to that of IM administered vaccination. Thus, these data support further investigation into STI-7264 and lymphatic mediated delivery using MuVaxx for SARS-CoV-2 and VoCs vaccines.

The identification of a vaccination candidate against COVID-19 providing protecting activity against emerging SARS-COV-2 variants remains challenging. Here, we report protection activity against a spectrum of SARS-COV-2 and variants by immunization with protein-based recombinant RBD-C-tag administered with aluminum-phosphate adjuvant intramuscularly. Immunization of C57BL/6 mice with RBD-C-tag resulted in the in vivo production of IgG antibodies recognizing the immune-critical spike protein of the SARS-COV-2 virus as well as the SARS-COV-2 variants alpha (“United Kingdom”), beta (“South Africa”), gamma (“Brazil/Japan”), and delta (“India”) as well as wt-spike protein. RBD-C-tag immunization led to a desired Th1 polarization of CD4 T cells producing IFNγ. Importantly, RBD-C-tag immunization educated IgG production delivers antibodies that exert neutralizing activity against the highly transmissible SARS-COV-2 virus strains “Washington”, “South Africa” (beta), and “India” (delta) as determined by conservative infection protection experiments in vitro. Hence, the protein-based recombinant RBD-C-tag is considered a promising vaccination candidate against COVID-19 and a broad range of emerging SARS-COV-2 virus variants.

As an essential enzyme of SARS-CoV-2, main protease (M

Chronic infection and cancer are associated with suppressed T cell responses in the presence of cognate antigen. Recent work identified memory-like CXCR5+ TCF1+ CD8+ T cells that sustain T cell responses during persistent infection and proliferate upon anti-PD1 treatment. Approaches to expand these cells are sought. We show that blockade of interferon type 1 (IFN-I) receptor leads to CXCR5+ CD8+ T cell expansion in an IL-27– and STAT1-dependent manner. IFNAR1 blockade promoted accelerated cell division and retention of TCF1 in virus-specific CD8+ T cells. We found that CD8+ T cell–intrinsic IL-27 signaling safeguards the ability of TCF1hi cells to maintain proliferation and avoid terminal differentiation or programmed cell death. Mechanistically, IL-27 endowed rapidly dividing cells with IRF1, a transcription factor that was required for sustained division in a cell-intrinsic manner. These findings reveal that IL-27 opposes IFN-I to uncouple effector differentiation from cell division and suggest that IL-27 signaling could be exploited to augment self-renewing T cells in chronic infections and cancer.

As an essential enzyme of SARS-CoV-2, the pathogen of COVID-19, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor with cellular potency. By coupling this toxicity alleviation with the expression of an MPro-eGFP fusion protein in a human cell host for straightforward characterization with fluorescent flow cytometry, we developed an effective method that allows bulk analysis of cellular potency of MPro inhibitors. In comparison to an antiviral assay in which MPro inhibitors may target host proteases or other processes in the SARS-CoV-2 life cycle to convene strong antiviral effects, this novel assay is more advantageous in providing precise cellular MPro inhibition information for assessment and optimization of MPro inhibitors. We used this assay to analyze 30 literature reported MPro inhibitors including MPI1-9 that were newly developed aldehyde-based reversible covalent inhibitors of MPro, GC376 and 11a that are two investigational drugs undergoing clinical trials for the treatment of COVID-19 patients in United States, boceprevir, calpain inhibitor II, calpain inhibitor XII, ebselen, bepridil that is an antianginal drug with potent anti-SARS-CoV-2 activity, and chloroquine and hydroxychloroquine that were previously shown to inhibit MPro. Our results showed that most inhibitors displayed cellular potency much weaker than their potency in direct inhibition of the enzyme. Many inhibitors exhibited weak or undetectable cellular potency up to 10 μM. On contrary to their strong antiviral effects, 11a, calpain inhibitor II, calpain XII, ebselen, and bepridil showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle to convene potent antiviral effects. characterization of these molecules on their antiviral mechanisms will likely reveal novel drug targets for COVID-19. Chloroquine and hydroxychloroquine showed close to undetectable cellular potency to inhibit MPro. Kinetic recharacterization of these two compounds rules out their possibility as MPro inhibitors. Our results also revealed that MPI5, 6, 7, and 8 have high cellular and antiviral potency with both IC50 and EC50 values respectively below 1 μM. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Given its strong cellular and antiviral potency, we cautiously suggest that MPI8 is ready for preclinical and clinical investigations for the treatment of COVID-19.

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets., Here, the authors perform repurposing screens of the ReFRAME drug library in two cell lines and identify inhibitors of SARS-CoV-2 infection. Antiviral activity of prodrug MK-4482 is confirmed in hamsters.

It is unclear whether individuals with enormous diversity in B cell receptor repertoires are consistently able to mount effective antibody responses against SARS-CoV-2. We analyzed antibody responses in a cohort of 55 convalescent patients and isolated 54 potent neutralizing monoclonal antibodies (mAbs). While most of the mAbs target the angiotensin-converting enzyme 2 (ACE2) binding surface on the receptor binding domain (RBD) of SARS-CoV-2 spike protein, mAb 47D1 binds only to one side of the receptor binding surface on the RBD. Neutralization by 47D1 is achieved independent of interfering RBD-ACE2 binding. A crystal structure of the mAb-RBD complex shows that the IF motif at the tip of 47D1 CDR H2 interacts with a hydrophobic pocket in the RBD. Diverse immunoglobulin gene usage and convergent epitope targeting characterize neutralizing antibody responses to SARS-CoV-2, suggesting that vaccines that effectively present the receptor binding site on the RBD will likely elicit neutralizing antibody responses in a large fraction of the population., Graphical abstract, Zhou et al. isolate 54 potent neutralizing antibodies from convalescent COVID-19 patients, revealing diverse immunoglobulin gene usage and convergent epitope targeting of neutralizing antibody responses to SARS-CoV-2. Vaccines that effectively present the RBD receptor binding site will likely elicit neutralizing antibody responses in a large fraction of the population.

Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and, importantly, as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a coronavirus disease 2019 (COVID-19)-convalescent donor that exhibits broad reactivity with human beta-coronaviruses (β-CoVs). Here, we showed that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 Å resolution and found that the peptide adopted a mainly helical structure. Conserved residues in β-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on β-CoV spike proteins for protective antibodies that may facilitate the development of pan-β-CoV vaccines.SUMMARYA human mAb isolated from a COVID-19 donor defines a protective cross-neutralizing epitope for pan-β-CoV vaccine design strategies

Type 1 interferon provides potent antiviral defense but unchecked IFN-I signaling can lead to pathology or immune suppression. The mechanisms by which IFN-I contributes to disease pathogenesis remain poorly understood. Here, using Usp18-deficient, USP18 enzymatic-inactive and Isg15-deficient mouse models, we report that lack of USP18 enzymatic function during persistent viral infection leads to severe immune pathology characterized by hematological disruptions, pulmonary cytokine amplification, lung vascular leakage and death. Lack of Usp18 in myeloid cells mimicked the pathological manifestations observed in Usp18-/- or Usp18C61A knock-in mice and required Isg15. Mechanistically, interrupting the enzymes that conjugate/deconjugate ISG15 led to accumulation of ISG15 which was accompanied by inflammatory neutrophil accumulation and lung pathology. Moreover, neutrophil depletion reversed pathological manifestations, morbidity and mortality in Usp18C61A mice. Our results suggest that dysregulated ISG15 signaling during viral infection can produce lethal immune pathology and could be a therapeutic target during severe viral infections with pulmonary pathological manifestations.

The sympathetic nervous system (SNS) contributes to immune balance by promoting anti-inflammatory B cells. However, whether B cells possess a self-regulating mechanism by which they modulate regulatory B cell (Breg) function is not well understood. In this study, we investigated the ability of B cells to synthesize catecholamines upon stimulation with different B cell activators. We found, that expression of the enzymes required to generate catecholamines, is upregulated by TLR9. TLR-9-specific expression of tyrosine hydroxylase (TH) correlated with upregulation of adrenergic receptors, enhanced IL-10 production, and with an overexpression of the co-inhibitory ligands PD-L1 and FasL. Moreover, concomitant stimulation of The sympathetic nervous system (SNS) contributes to immune balance by promoting anti-inflammatory B cells. However, whether B cells possess a self-regulating mechanism by which they modulate regulatory B cell (Breg) function is not well understood. In this study, we investigated the ability of B cells to synthesize catecholamines upon stimulation with different B cell activators. We found, that expression of the enzymes required to generate catecholamines, is upregulated by TLR9. TLR-9-specific expression of tyrosine hydroxylase (TH) correlated with upregulation of adrenergic receptors, enhanced IL-10 production, and with an overexpression of the co-inhibitory ligands PD-L1 and FasL. Moreover, concomitant stimulation of beta 1-3-adrenergic receptors together with a BCR/TLR9 stimulus enhances the anti-inflammatory potential of Bregs to suppress CD4 T cells, a crucial population in the pathogenesis of autoimmune diseases, like rheumatoid arthritis. In conclusion, our data show that B cells possess autonomous mechanisms to modulate their regulatory function. These findings help to better understand the function of Bregs in autoimmune diseases and the interplay of sympathetic nervous system and B cell function. Beta 1-3-adrenergic receptors together with a BCR/TLR9 stimulus enhances the anti-inflammatory potential of Bregs to suppress CD4 T cells, a crucial population in the pathogenesis of autoimmune diseases, like rheumatoid arthritis. In conclusion, our data show that B cells possess autonomous mechanisms to modulate their regulatory function. These findings help to better understand the function of Bregs in autoimmune diseases and the interplay of sympathetic nervous system and B cell function.

Cytokine storm during respiratory viral infection is an indicator of disease severity and poor prognosis. Type 1 interferon (IFN-I) production and signaling has been reported to be causal in cytokine storm-associated pathology in several respiratory viral infections, however, the mechanisms by which IFN-I promotes disease pathogenesis remain poorly understood. Here, using Usp18-deficient, USP18 enzymatic-inactive and Isg15-deficient mouse models, we report that lack of deISGylation during persistent viral infection leads to severe immune pathology characterized by hematological disruptions, cytokine amplification, lung vascular leakage and death. This pathology requires T cells but not T cell-intrinsic deletion of Usp18. However, lack of Usp18 in myeloid cells mimicked the pathological manifestations observed in Usp18-/- or Usp18C61A mice which were dependent on Isg15. We further mechanistically demonstrate that interrupting the ISGylation/deISGylation circuit increases extracellular levels of ISG15 which is accompanied by inflammatory neutrophil accumulation to the lung. Importantly, neutrophil depletion reversed morbidity and mortality in Usp18C61A mice. In summary, we reveal that the enzymatic function of Usp18 is crucial for regulating extracellular release of ISG15. This is accompanied by altered neutrophil differentiation, cytokine amplification and mortality following persistent viral infection. Moreover, our results suggest that extracellular ISG15 may drive the inflammatory pathology observed and could be both a prospective predictor of disease outcome and a therapeutic target during severe respiratory viral infections.

IFNAR1 signaling in NK cells compromises cellular and humoral immune responses to hamper virus control., Inhibition of type 1 interferon (IFN-I) signaling promotes the control of persistent virus infection, but the underlying mechanisms remain poorly understood. Here, we report that genetic ablation of Ifnar1 specifically in natural killer (NK) cells led to elevated numbers of T follicular helper cells, germinal center B cells, and plasma cells and improved antiviral T cell function, resulting in hastened virus clearance that was comparable to IFNAR1 neutralizing antibody treatment. Antigen-specific B cells and antiviral antibodies were essential for the accelerated control of LCMV Cl13 infection following IFNAR1 blockade. IFNAR1 signaling in NK cells promoted NK cell function and general killing of antigen-specific CD4 and CD8 T cells. Therefore, inhibition of IFN-I signaling in NK cells enhances CD4 and CD8 T cell responses, promotes humoral immune responses, and thereby facilitates the control of persistent virus infection.

Combination immunotherapy (CIT) is currently applied as a treatment for different cancers and is proposed as a cure strategy for chronic viral infections. Whether such therapies are efficient during an acute infection remains elusive. To address this, inhibitory receptors were blocked and regulatory T cells depleted in acutely Friend retrovirus-infected mice. CIT resulted in a dramatic expansion of cytotoxic CD4+ and CD8+ T cells and a subsequent reduction in viral loads. Despite limited viral replication, mice developed fatal immunopathology after CIT. The pathology was most severe in the gastrointestinal tract and was mediated by granzyme B producing CD4+ and CD8+ T cells. A similar post-CIT pathology during acute Influenza virus infection of mice was observed, which could be prevented by vaccination. Melanoma patients who developed immune-related adverse events under immune checkpoint CIT also presented with expanded granzyme-expressing CD4+ and CD8+ T cell populations. Our data suggest that acute infections may induce immunopathology in patients treated with CIT, and that effective measures for infection prevention should be applied., Author summary Combination immunotherapy (CIT) directed against checkpoint mechanisms has been approved for the therapy of cancers and is proposed for the treatment of chronic infections. In cancer therapy patients often develop severe immunopathology under CIT. Here we show that acute viral infections (Friend retrovirus and Influenza virus) posed a significant threat during CIT in mice. The strong activation of cytotoxic T cells after an acute viral infection seemed to lack couterregulation during CIT, which resulted in lethal immunopathology. In case of an Influenza virus infection this could be prevented by vaccination prior to CIT. The expansion of CD4+ and CD8+ T cells expressing cytotoxic molecules were also observed in melanoma patients treated with two checkpoint blockers. Thus, these patients might also be at risk of developing severe immunopathology during an otherwise harmless acute viral infection. This should to be taken into account when cancer patients are undergoing CIT.

The TAM family of receptor tyrosine kinases (MERTK, AXL, and TYRO3) are expressed on DCs and macrophages, and they serve a critical role in efferocytosis, by which apoptotic bodies are cleared by engulfment. Their activation drives phagocytic activity concomitant to promoting an immuno-suppressive, tolerogenic state in DCs and the M2 polarization of macrophages. This dual signaling mechanism allows for the clearance of cells undergoing non-pathologic apoptotic turnover without inappropriately breaking self-tolerance and driving auto-immunity. Unlike most immuno-therapy targets, the KOs of the TAM kinases, PD-1, and CTLA-4 share common auto-immune disease phenotypes such as splenomegaly, immune infiltration of the liver, arthritis, and lupus. Furthermore, viruses have evolved to promote PD-1/CTLA-4 signaling to suppress the anti-viral immune response, and they similarly target the TAM receptors via apoptotic mimicry to reduce the inflammatory response. This confluence of immune phenotypes provided the biological rationale for the discovery of PF-5807/ARRY-067, a potent and selective inhibitor of MERTK/AXL as a DC-activating immuno-oncology therapeutic. Based on the IC50 for inhibition of phospho-MERTK/AXL in cell-based assays, our mouse PK and PK/PD studies determined that 50 mg/kg PF-5807 provided IC90 plasma coverage for 12 hours. This PK profile supported a twice-daily dosing schedule for maintaining continuous suppression of MERTK and AXL, which was used for mechanistic and efficacy studies in mouse models. To investigate DC and T cell responses to PF-5807, mice were adoptively-transferred with OT-I T cells and challenged with ovalbumin immunization; flow cytometry analysis indicated that PF-5807 treatment increased the fraction of activated, Class II MHC-positive DCs, as well as cross-primed IFNγ+ TNFα+ OT-I T cells. Also, we found that cytotoxic T lymphocytes were necessary for tumor control in the E0771 mouse syngeneic tumor model, as depletion of CD8+ T cells abrogated all activity by PF-5807 in inhibiting tumor growth. This anti-tumor activity was further demonstrated in the MC-38 and EMT6 tumor models, where single agent PF-5807 effected 57-88% TGI and a 13-38% cure rate, while combination with PD-1 blockade drove 99-100% TGI and a 75-100% cure rate. Finally, these complete responders rejected tumor re-challenge without further treatment, resulting in extended survival and durable cures that corroborate the development of immunologic memory. Therefore, we find that PF-5807 activates DCs to cross-prime CD8+ T cells, which are necessary for tumor control and are likely responsible for the establishment of tumor-specific memory T cells. PF-5807 is currently in an ongoing Phase I clinical trial in patients with advanced or metastatic solid tumors, with the intent to explore combination with anti-PD-1/anti-PD-L1 antibodies. Citation Format: Jim Wong, Jacqueline Harrison, Karyn Bouhana, Nhan Nguyen, Namir Shaabani, Vincent F. Vartabedian, LouAnn Cable, Robert Rieger, Lisa Pieti Opie, Shelley Allen, Adam Cook, Joshua Dahlke, Ronald Hinklin, David Chantry, Shannon Winski, John Teijaro. The potent and selective MERTK/AXL inhibitor PF-5807/ARRY-067 activates dendritic cells to cross-prime CD8+ T cells for anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1735.

Background: Bile acids (BAs) are steroid molecules that are synthesized in the liver. In addition to their important role as a surfactant in solubilizing lipids and promoting the absorption of lipids in the gastrointestinal tract, they act as inflammagens. The role of BAs and their receptor farnesoid X receptor (FXR) during viral infection has not been studied in detail. Methods: By using FXR-deficient mice, we investigated the role of bile acid receptor FXR during infection with lymphocytic choriomeningitis virus (LCMV). The importance of FXR in inducing IFN-I and monocytes proliferation were investigated and viral titers and T cell exhaustion were analyzed at different time points. Results: This study shows that controlled levels of BAs activate FXR in hepatocytes and FXR in response upregulates the production of type I interferon. In turn, FXR maintains BAs within a balanced range to inhibit their toxic effects. The absence of FXR results in high levels of BAs, which inhibit the proliferation of monocytes and result in a defect in viral elimination, consequently leading to T cell exhaustion. Conclusion: We found that FXR contributes to IFN-I production in hepatocytes and balances BA levels to inhibit their toxic effects on monocytes.

CXCR5+ TCF1+ CD8+ T cells sustain responses during persistent viral infection and mediate the proliferative burst following anti-PD1 treatment. Huang et al. show that IL-27 supports rapid division of these cells by competing with type 1 interferon for STAT1, driving IRF1 expression and preventing cell death., Chronic infection and cancer are associated with suppressed T cell responses in the presence of cognate antigen. Recent work identified memory-like CXCR5+ TCF1+ CD8+ T cells that sustain T cell responses during persistent infection and proliferate upon anti-PD1 treatment. Approaches to expand these cells are sought. We show that blockade of interferon type 1 (IFN-I) receptor leads to CXCR5+ CD8+ T cell expansion in an IL-27– and STAT1-dependent manner. IFNAR1 blockade promoted accelerated cell division and retention of TCF1 in virus-specific CD8+ T cells. We found that CD8+ T cell–intrinsic IL-27 signaling safeguards the ability of TCF1hi cells to maintain proliferation and avoid terminal differentiation or programmed cell death. Mechanistically, IL-27 endowed rapidly dividing cells with IRF1, a transcription factor that was required for sustained division in a cell-intrinsic manner. These findings reveal that IL-27 opposes IFN-I to uncouple effector differentiation from cell division and suggest that IL-27 signaling could be exploited to augment self-renewing T cells in chronic infections and cancer., Graphical Abstract

Background: Graft versus host disease (GvHD) occurs in 20% of cases with patients having an MHC I matched bone marrow transplantation (BMT). Mechanisms causing this disease remain to be studied. Methods: Here we used a CD8+ T cell transgenic mouse line (P14/CD45.1+) and transgenic DEE mice bearing ubiquitously the glycoprotein 33-41 (GP33) antigen derived from the major lymphocytic choriomeningitis virus (LCMV) epitope to study mechanisms of tolerance in anti-host reactive CD8+ T cells after BMT. Results: We found that anti-host reactive CD8+ T cells (P14 T cells) were not negatively selected in the thymus and that they were present in wild type (WT) recipient mice as well as in DEE recipient mice. Anti-host reactive CD8+ T cells ignored the GP33 antigen expressed ubiquitously by host cells but they could be activated ex vivo via LCMV-infection. Lipopolysaccharides (LPS) induced transient cell damage in DEE mice bearing anti-host reactive CD8+ T cells after BMT, suggesting that induction of host inflammatory response could break antigen ignorance. Introducing the GP33 antigen into BM cells led to deletion of anti-host reactive CD8+ T cells. Conclusion: We found that after BMT anti-host reactive CD8+ T cells ignored host antigen in recipients and that they were only deleted when host antigen was present in hematopoietic cells. Moreover, LPS-induced immune activation contributed to induction of alloreactivity of anti-host reactive CD8+ T cells after BMT.

ISG15 is one of the strongest type I interferon (IFN-I) stimulated genes (ISGs) which is conjugated to proteins in an enzymatic process termed ISGylation. Protein ISGylation is subsequently reversed by the enzyme Usp18 which acts in a de-ubiqutinase fashion to remove SIG15 from target proteins. Many proteins are ISGylated following IFN-I signaling however, immunological studies have been limited to the anti-viral properties of ISG15 conjugation while its regulation of innate and adaptive immune responses has been poorly studied. Here using Usp18−/− and enzymatically inactive (Usp18C61A/C61A Knock-in) mouse models, we show that hyper-ISGylation during chronic viral infection leads to severe immune pathology characterized by hematological disruptions, cytokine amplification, lung vascular leakage and death. The pathology associated with chronic infection required T cells but not T cell-intrinsic Usp18 expression. We reveal that lack of Usp18 in LysM+ cells reproduced the pathological manifestations observed in Usp18−/− or Usp18C61A/C61A mice. Using single cell sequencing, we observed that hyper-ISGylation during chronic infection altered neutrophil maturation and promoted an inflammatory neutrophil lung infiltrate. Importantly, neutrophil depletion reversed morbidity and mortality in Usp18C61A/C61A mice. In summary, we reveal Usp18’s enzymatic function is crucial for regulating inflammatory neutrophil differentiation and preventing immune hyperactivation during chronic viral infection. Moreover, our results suggest that hyper-ISGylation may drive the inflammatory pathology observed in Usp18-null patients and indicate that regulation of ISG15 expression or conjugation could lessen disease.

To prime T cells to fight cancer, dendritic cells (DCs) require Type I Interferon (IFN-I) signaling. IFN-I signaling induces the transcription and translation of Interferon Stimulated Genes (ISGs), including strong induction of Usp18, which dampens further IFN-I signaling. We hypothesized that increasing IFN-I signals to DCs in vivo via conditional Usp18 deletion would enhance the ability of DCs to prime T cells, augmenting their anti-tumor activity. To test this, we used Usp18fl/fl x CD11c-cre mice and the syngeneic B16F10 melanoma model. Surprisingly, tumors grew more quickly in Usp18fl/fl x CD11c-cre mice, and intratumoral T cells were less functional post-priming by Usp18-deficient DCs. This functional decrease was coupled with an accelerated effector differentiation marked by early loss of the stem cell-like factor TCF-1. These data suggest that Usp18-deficient DCs expedite terminal effector T cell differentiation, resulting in suppressed effector functionality at later time points. Consistent with this T cell dysfunction, intratumoral STING agonism was less effective at controlling tumors in Usp18fl/fl x CD11c-Cre mice than Usp18fl/fl littermate controls. To understand the molecular drivers of these changes, we profiled DCs with tandem mass tag (TMT) mass spectrometry. We found that Usp18-deficient DCs exhibit deficiencies in mitochondrial function and cysteine biosynthesis, potentially driving their altered ability to prime T cells. Overall, this work highlights the importance of stringent IFN-I signaling regulation for mounting optimal immune responses to cancer. Our findings also identify DC-intrinsic Usp18 expression as a potential biomarker for predicting patient responsiveness to STING agonist therapy.

Type I interferon (IFN-I) signaling paradoxically impairs host immune responses during many primary and secondary bacterial infections. Lack of IFN-I receptor reduces bacterial replication and/or bacterial persistence during infection with several bacteria. However, the mechanisms that mediate the adverse IFN-I effect are incompletely understood. Here, we show that Usp18, an interferon-stimulated gene that negatively regulates IFN-I signaling, is primarily responsible for the deleterious effect of IFN-I signaling during infection of mice with Listeria monocytogenes or Staphylococcus aureus. Mechanistically, USP18 promoted bacterial replication by inhibiting antibacterial tumor necrosis factor-α (TNF-α) signaling. Deleting IFNAR1 or USP18 in CD11c-Cre(+) cells similarly reduced bacterial titers in multiple organs and enhanced survival. Our results demonstrate that inhibiting USP18 function can promote control of primary and secondary bacterial infection by enhancing the antibacterial effect of TNF-α, which correlates with induction of reactive oxygen species (ROS). These findings suggest that USP18 could be targeted therapeutically in patients to ameliorate disease caused by serious bacterial infections.

CD169 + macrophages are a unique subset of macrophages known for their special location in different organs and their ability to support viral replication for the presentation and activation of adaptive immune cells. Simultaneously, they restrict viral replication in other cells through production of IFN-I and balance T cell activation through upregulation of PD-L1 to prevent immunopathology.

Innate immune activation is essential to mount an effective antiviral response and to prime adaptive immunity. Although a crucial role of CD169+ cells during vesicular stomatitis virus (VSV) infections is increasingly recognized, factors regulating CD169+ cells during viral infections remain unclear. Here, we show that tumor necrosis factor is produced by CD11b+ Ly6C+ Ly6G+ cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169+ cells and in reduced type I interferon (IFN-I) production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nuclei of CD169+ cells; this translocation was inhibited when the paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and development of severe disease. These findings indicate that TNF mediates the maintenance of CD169+ cells and innate and adaptive immune activation during VSV infection. Fil: Shinde, Prashant V.. Heinrich Heine University; Alemania Fil: Xu, Haifeng C.. Heinrich Heine University; Alemania Fil: Maney, Sathish Kumar. Heinrich Heine University; Alemania Fil: Kloetgen, Andreas. Heinrich Heine University; Alemania Fil: Namineni, Sukumar. Helmholtz Zentrum Munich; Alemania Fil: Zhuang, Yuan. Heinrich Heine University; Alemania Fil: Honke, Nadine. Heinrich Heine University; Alemania Fil: Shaabani, Namir. The Scripps Research Institute; Estados Unidos Fil: Bellora, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales.; Argentina Fil: Doerrenberg, Mareike. Heinrich Heine University; Alemania Fil: Trilling, Mirko. Universitat Essen; Alemania Fil: Pozdeev, Vitaly I.. Heinrich Heine University; Alemania Fil: van Rooijen, Nico. Department Of Cell Biology, Vrije University; Países Bajos Fil: Scheu, Stefanie. Heinrich Heine University; Alemania Fil: Pfeffer, Klaus. Heinrich Heine University; Alemania Fil: Crocker, Paul R.. University of Dundee; Reino Unido Fil: Tanaka, Masato. Tokyo University. Laboratory Of Immune Regulation; Japón Fil: Duggimpudi, Sujitha. Heinrich Heine University; Alemania Fil: Knolle, Percy. Helmholtz Zentrum Munich; Alemania Fil: Heikenwalder, Mathias. Helmholtz Zentrum Munich; Alemania Fil: Ruland, Jürgen. Universitat Technical Zu Munich; Alemania Fil: Mak, Tak W.. University Health Network; Canadá Fil: Brenner, Dirk. University of Southern Denmark; Dinamarca Fil: Pandyra, Aleksandra A.. Universitat Essen; Alemania Fil: Hoell, Jessica I.. Heinrich Heine University; Alemania Fil: Borkhardt, Arndt. Heinrich Heine University; Alemania Fil: Häussinger, Dieter. Heinrich Heine University; Alemania Fil: Lang, Karl S.. Universitat Essen; Alemania Fil: Lang, Philipp A.. Heinrich Heine University; Alemania