The Kinase IKKß Regulates a STING- and NF-κB-Dependent Antiviral Response Pathway in Drosophila.

Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. Here, we showed that two components of the IMD pathway, the kinase dIKKß and the transcription factor Relish, were required to control infection by two picorna-like viruses. We identified a set of genes induced by viral infection and regulated by dIKKß and Relish, which included an ortholog of STING. We showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKß to regulate expression of Nazo, an antiviral factor. Our data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity.

Two cGAS-like receptors induce antiviral immunity in Drosophila.

In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases that is present in both prokaryotes1 and eukaryotes2-5. In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections6-8. Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. We show that cGLR1 and cGLR2 activate Sting- and NF-κB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3'2'-cGAMP, whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2'-cGAMP in response to an as-yet-unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2'3'-cGAMP.

Length dependent activation of OAS proteins by dsRNA.

The oligoadenylate synthetase (OAS) family of enzymes are interferon-inducible antiviral proteins, which synthesize the secondary messenger 2'-5'-linked oligoadenosine (2-5A) in response to viral infection. The production of 2-5As induces RNA decay within the infected cells, thereby effectively preventing further viral replication. OAS shares structural similarity as well as the enzymatic mechanism with a different antiviral protein, cyclic GMP-AMP synthase (cGAS), but OAS is activated by dsRNA whereas cGAS is activated by dsDNA. Here, we have studied the structural requirement for the dsRNA activating OAS1 and OAS3, and compared it to recent studies on cGAS. We find that both OAS1 and OAS3, like cGAS, achieve their maximum activity with dsRNA molecules that are substantial longer than what one monomer of the enzyme can interact with. One molecule of OAS1 can cover approximately 18-20 base pairs of dsRNA, which is just short of two turns of a helix. However, RNAs of this length gave a very limited activity and the length dependency was even more pronounced for OAS3. Our data suggest that the OAS enzymes evolved to recognize long dsRNA as virally derived PAMPs, and that the length of the dsRNA is an important factor in discriminating self from non-self. Several structures of OAS1 bound to short dsRNAs exist, but our data show that OAS can only achieve minimal activity with these short activators (approximately 7-8% of maximal activity) and it is thus possible that these structures do not reveal the fully activated state of the OAS enzymes.

cGAS is activated by DNA in a length-dependent manner.

Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP-AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length-dependent manner. This is observed over a wide length-span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length. Importantly, in vitro studies reveal that long DNA activates recombinant human cGAS more efficiently than short DNA, showing that length-dependent DNA recognition is an intrinsic property of cGAS independent of accessory proteins. Collectively, this work identifies long DNA as the molecular entity stimulating the cGAS pathway upon cytosolic DNA challenge such as viral infections.

Redirector of Vaccine-induced Effector Responses (RoVER) for specific killing of cellular targets.

BACKGROUND: In individuals with malignancy or HIV-1 infection, antigen-specific cytotoxic T lymphocytes (CTLs) often display an exhausted phenotype with impaired capacity to eliminate the disease. Existing cell-based immunotherapy strategies are often limited by the requirement for adoptive transfer of CTLs. We have developed an immunotherapy technology in which potent CTL responses are generated in vivo by vaccination and redirected to eliminate target cells using a bispecific Redirector of Vaccine-induced Effector Responses (RoVER). METHODS: Following Yellow fever (YF) 17D vaccination of 51 healthy volunteers (NCT04083430), single-epitope YF-specific CTL responses were quantified by tetramer staining and multi-parameter flow cytometry. RoVER-mediated redirection of YF-specific CTLs to kill antigen-expressing Raji-Env cells, autologous CD19+ B cells or CD4+ T cells infected in vitro with a full-length HIV-1-eGFP was assessed in cell killing assays. Moreover, secreted IFN-γ, granzyme B, and TNF-α were analyzed by mesoscale multiplex assays. FINDINGS: YF-17D vaccination induced strong epitope-specific CTL responses in the study participants. In cell killing assays, RoVER-mediated redirection of YF-specific CTLs to autologous CD19+ B cells or HIV-1-infected CD4+ cells resulted in 58% and 53% killing at effector to target ratio 1:1, respectively. INTERPRETATION: We have developed an immunotherapy technology in which epitope-specific CTLs induced by vaccination can be redirected to kill antigen-expressing target cells by RoVER linking. The RoVER technology is highly specific and can be adapted to recognize various cell surface antigens. Importantly, this technology obviates the need for adoptive transfer of CTLs. FUNDING: This work was funded by the Novo Nordisk Foundation (Hallas Møller NNF10OC0054577).

SARS-CoV-2 elicits robust adaptive immune responses regardless of disease severity.

BACKGROUND: The SARS-CoV-2 pandemic currently prevails worldwide. To understand the immunological signature of SARS-CoV-2 infections and aid the search and evaluation of new treatment modalities and vaccines, comprehensive characterization of adaptive immune responses towards SARS-CoV-2 is needed. METHODS: We included 203 recovered SARS-CoV-2 infected patients in Denmark between April 3rd and July 9th 2020, at least 14 days after COVID-19 symptom recovery. The participants had experienced a range of disease severities from asymptomatic to severe. We collected plasma, serum and PBMC's for analysis of SARS-CoV-2 specific antibody response by Meso Scale analysis including other coronavirus strains, ACE2 competition, IgA ELISA, pseudovirus neutralization capacity, and dextramer flow cytometry analysis of CD8+ T cells. The immunological outcomes were compared amongst severity groups within the cohort, and 10 pre-pandemic SARS-CoV-2 negative controls. FINDINGS: We report broad serological profiles within the cohort, detecting antibody binding to other human coronaviruses. 202(>99%) participants had SARS-CoV-2 specific antibodies, with SARS-CoV-2 neutralization and spike-ACE2 receptor interaction blocking observed in 193(95%) individuals. A significant positive correlation (r=0.7804) between spike-ACE2 blocking antibody titers and neutralization potency was observed. Further, SARS-CoV-2 specific CD8+ T-cell responses were clear and quantifiable in 95 of 106(90%) HLA-A2+ individuals. INTERPRETATION: The viral surface spike protein was identified as the dominant target for both neutralizing antibodies and CD8+ T-cell responses. Overall, the majority of patients had robust adaptive immune responses, regardless of their disease severity. FUNDING: This study was supported by the Danish Ministry for Research and Education (grant# 0238-00001B) and The Danish Innovation Fund (grant# 0208-00018B).

2'3'-cGAMP triggers a STING- and NF-κB-dependent broad antiviral response in Drosophila.

We previously reported that an ortholog of STING regulates infection by picorna-like viruses in Drosophila In mammals, STING is activated by the cyclic dinucleotide 2'3'-cGAMP produced by cGAS, which acts as a receptor for cytosolic DNA. Here, we showed that injection of flies with 2'3'-cGAMP induced the expression of dSTING-regulated genes. Coinjection of 2'3'-cGAMP with a panel of RNA or DNA viruses resulted in substantially reduced viral replication. This 2'3'-cGAMP-mediated protection was still observed in flies with mutations in Atg7 and AGO2, genes that encode key components of the autophagy and small interfering RNA pathways, respectively. By contrast, this protection was abrogated in flies with mutations in the gene encoding the NF-κB transcription factor Relish. Transcriptomic analysis of 2'3'-cGAMP-injected flies revealed a complex response pattern in which genes were rapidly induced, induced after a delay, or induced in a sustained manner. Our results reveal that dSTING regulates an NF-κB-dependent antiviral program that predates the emergence of interferons in vertebrates.

A Highly Sensitive Anion Exchange Chromatography Method forMeasuring cGAS Activity in vitro.

Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor (PRR) that senses double stranded DNA (dsDNA) in the cytosol and this leads to the activation of stimulator of interferon genes (STING) via the secondary messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP). STING then recruits TANK binding kinase 1 (TBK-1) and this complex can phosphorylate and activate interferon regulatory factor 3 (IRF3) leading to the induction of type I interferons and other antiviral genes. The cGAS:DNA complex catalyzes the synthesis of 2'3'-cGAMP and the purpose of the protocol presented here is to measure the in vitro activity of purified cGAS in the presence of dsDNA. The protocol was developed to elucidate the relationship between dsDNA length and the level of cGAS activity. The method involves an in vitro reaction with low concentrations of cGAS and dsDNA followed by quantification of the reaction product using anion exchange chromatography. The low concentrations of cGAS and dsDNA and the high sensitivity of this assay is a key advantage when comparing different DNA fragments' ability to activate cGAS.