Stimulator of Interferon Genes (STING) is a critical component of host innate immune defense but can contribute to chronic autoimmune or autoinflammatory disease. Once activated, the cyclic guanosine ...monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS)-STING pathway induces both type I interferon (IFN) expression and nuclear factor-κB (NF-κB)-mediated cytokine production. Currently, these two signaling arms are thought to be mediated by a single upstream kinase, TANK-binding kinase 1 (TBK1). Here, using genetic and pharmacological approaches, we show that TBK1 alone is dispensable for STING-induced NF-κB responses in human and mouse immune cells, as well as in vivo. We further demonstrate that TBK1 acts redundantly with IκB kinase ε (IKKε) to drive NF-κB upon STING activation. Interestingly, we show that activation of IFN regulatory factor 3 (IRF3) is highly dependent on TBK1 kinase activity, whereas NF-κB is significantly less sensitive to TBK1/IKKε kinase inhibition. Our work redefines signaling events downstream of cGAS-STING. Our findings further suggest that cGAS-STING will need to be targeted directly to effectively ameliorate the inflammation underpinning disorders associated with STING hyperactivity.
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•TBK1 is dispensable for NF-κB activation downstream of STING in vitro and in vivo•TBK1 and IKKε act redundantly to elicit STING-induced NF-κB activation•STING-NF-κB is less sensitive to TBK1/IKKε kinase inhibition than type I IFN•TAK1 and IKK complexes are required for STING-mediated NF-κB responses
Activation of NF-κB via STING is considered to be exclusively dependent on TBK1. Balka et al. now show that, although TBK1 and its kinase activity are critical for IRF3 activation and type I IFNs, it is dispensable for NF-κB. Instead, TBK1 and IKKε act redundantly to mediate STING-induced NF-κB responses.
Monogenic autoinflammatory disorders are characterized by dysregulation of the innate immune system, for example by gain-of-function mutations in inflammasome-forming proteins, such as NOD-like ...receptor family CARD-containing 4 protein (NLRC4).
Here we investigate the mechanism by which a novel mutation in the leucine-rich repeat (LRR) domain of NLRC4 (c.G1965C, p.W655C) contributes to autoinflammatory disease. Methods: We studied 2 unrelated patients with early-onset macrophage activation syndrome harboring the same de novo mutation in NLRC4. In vitro inflammasome complex formation was quantified by using flow cytometric analysis of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 techniques and lentiviral transduction were used to generate THP-1 cells with either wild-type or mutant NLRC4 cDNA. Cell death and release of IL-1β/IL-18 were quantified by using flow cytometry and ELISA, respectively.
The p.W655C NLRC4 mutation caused increased ASC speck formation, caspase-1–dependent cell death, and IL-1β/IL-18 production. ASC contributed to p.W655C NLRC4–mediated cytokine release but not cell death. Mutation of p.W655 activated the NLRC4 inflammasome complex by engaging with 2 interfaces on the opposing LRR domain of the oligomer. One key set of residues (p.D1010, p.D1011, p.L1012, and p.I1015) participated in LRR-LRR oligomerization when triggered by mutant NLRC4 or type 3 secretion system effector (PrgI) stimulation of the NLRC4 inflammasome complex.
This is the first report of a mutation in the LRR domain of NLRC4 causing autoinflammatory disease. c.G1965C/p.W655C NLRC4 increased inflammasome activation in vitro. Data generated from various NLRC4 mutations provides evidence that the LRR-LRR interface has an important and previously unrecognized role in oligomerization of the NLRC4 inflammasome complex.
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Mutations in the gene encoding stimulator of interferon genes (STING) underlie a type I interferon (IFN) associated disease, STING-associated vasculopathy with onset in infancy (SAVI). Patients ...suffer cutaneous vasculopathy and interstitial lung disease, but are not known to suffer life-threatening infection.
We describe a child who presented with
pneumonia in early life, from which he recovered. He went on to suffer failure to thrive, developmental delay, livedo reticularis, and vesicular rash, but without cutaneous vasculitis, and with normal C-reactive protein and erythrocyte sedimentation rates. At 3 years of age, he developed life-threatening pulmonary hypertension.
Whole genome sequencing (WGS) was performed using the Illumina HiSeqX10 platform and the Seave platform was used for bioinformatic analysis. mRNA expression of IFN-stimulated genes and inflammatory cytokines from peripheral blood mononuclear cells was determined by quantitative polymerase chain reaction. Luciferase assay was used to model IFNβ and NF-κB activity
.
WGS revealed a
mutation p.Arg284Ser in STING at an amino acid previously associated with SAVI. Although this mutation did not fall in the dimerization domain (DD), mRNA analysis revealed constitutive IFN-gene activation consistent with an interferonopathy, which correlated to STING activation
. The patient was treated with corticosteroids and the JAK inhibitor Ruxolitinib, resulting in a rapid improvement of pulmonary hypertension, general well-being, and resolution of the IFN gene signature. However, he did go on to evolve a nasal septal erosion suggesting incomplete control of disease.
This case provides molecular evidence to support the p.Arg284Ser variant in STING exerting pathogenicity through a gain-of-function mechanism. The lack of cutaneous vasculitis or elevated systemic inflammatory markers, and the occurrence of an opportunistic infection are notable, and raise the possibility that variants outside the STING DD may potentially manifest with an atypical SAVI phenotype. Nevertheless, there was an objective clinical improvement in response to JAK inhibition.
Purpose
NLRC4-associated autoinflammatory disease (NLRC4-AID) is an autosomal dominant condition presenting with a range of clinical manifestations which can include macrophage activation syndrome ...(MAS) and severe enterocolitis. We now report the first homozygous mutation in
NLRC4
(c.478G > A, p.A160T) causing autoinflammatory disease with immune dysregulation and find that heterozygous carriers in the general population are at increased risk of developing ulcerative colitis.
Methods
Circulating immune cells and inflammatory markers were profiled and historical clinical data interrogated. DNA was extracted and sequenced using standard procedures. Inflammasome activation assays for ASC speck formation, pyroptosis, and IL-1β/IL-18 secretion confirmed pathogenicity of the mutation in vitro. Genome-wide association of NLRC4 (A160T) with ulcerative colitis was examined using data from the IBD exomes portal.
Results
A 60-year-old Brazilian female patient was evaluated for recurrent episodes of systemic inflammation from six months of age. Episodes were characterized by recurrent low-grade fever, chills, oral ulceration, uveitis, arthralgia, and abdominal pain, followed by diarrhea with mucus and variable skin rash. High doses of corticosteroids were somewhat effective in controlling disease and anti-IL-1β therapy partially controlled symptoms. While on treatment, serum IL-1β and IL-18 levels remained elevated. Genetic investigations identified a homozygous mutation in
NLRC4
(A160T), inherited in a recessive fashion. Increased ASC speck formation and IL-1β/IL-18 secretion confirmed pathogenicity when NLRC4 (A160T) was analyzed in human cell lines. This allele is significantly enriched in patients with ulcerative colitis: OR 2.546 (95% 1.778–3.644),
P
= 0.01305.
Conclusion
NLRC4 (A160T) can either cause recessively inherited autoinflammation and immune dysregulation, or function as a heterozygous risk factor for the development of ulcerative colitis.
Activation of cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in antiviral responses to many DNA viruses. Sensing of cytosolic DNA by cGAS results in synthesis of the endogenous second ...messenger cGAMP that activates stimulator of interferon genes (STING) in infected cells. Critically, cGAMP can also propagate antiviral responses to uninfected cells through intercellular transfer, although the modalities of this transfer between epithelial and immune cells remain poorly defined. We demonstrate here that cGAMP-producing epithelial cells can transactivate STING in cocultured macrophages through direct cGAMP transfer. cGAMP transfer was reliant upon connexin expression by epithelial cells and pharmacological inhibition of connexins blunted
-dependent transactivation of the macrophage compartment. Macrophage transactivation by cGAMP contributed to a positive-feedback loop amplifying antiviral responses, significantly protecting uninfected epithelial cells against viral infection. Collectively, our findings constitute the first direct evidence of a connexin-dependent cGAMP transfer to macrophages by epithelial cells, to amplify antiviral responses.
Recent studies suggest that extracellular cGAMP can be taken up by macrophages to engage STING through several mechanisms. Our work demonstrates that connexin-dependent communication between epithelial cells and macrophages plays a significant role in the amplification of antiviral responses mediated by cGAMP and suggests that pharmacological strategies aimed at modulating connexins may have therapeutic applications to control antiviral responses in humans.
The past two decades have seen an exponential increase in the number of monogenic autoinflammatory disorders described, coinciding with improved genetic sequencing techniques. This group of disorders ...has evolved to be heterogeneous and certainly more complex than the original four ‘periodic fever syndromes’ caused by innate immune over‐activation. This review aims to provide an update on the classic periodic fever syndromes as well as introducing the broadening spectrum of clinical features seen in more recently described conditions.
The objective was to review COVID-19 vaccine allergy advice and guidance requests received and assess the impact of advice outcome on vaccination outcome.
A retrospective analysis of requests for ...advice and guidance regarding COVID-19 vaccine allergy was completed using an electronic referral system from February 2021 to January 2022.
A total of 1265 independent patient requests for advice were received from primary care. Full vaccination information was available on 1210 patients who were included in the analysis.
We evaluated the specific outcome of request for advice (written advice versus allergy consultation), rate of vaccination, vaccination combinations, and tolerance of vaccination.
Of the 1210 patients included, 959 (79%) were female. Eight hundred and ninety-six (74%) requests were managed with written advice only and of these 675 (75%) patients went on to be vaccinated. Overall, 891 (74%) of the population were vaccinated with 2 or more doses.
Two hundred and nineteen patient consultations were undertaken with 109 (50%) prior to the first vaccination. Forty-nine (45%) consultations prior to vaccination were undertaken due to a label of anaphylaxis to vaccination in the past. Vaccination was recommended for all patients, and 78 (72%) of these received a first dose. Eight of these patients (10%) had symptoms within 1 h of vaccine administration.
One hundred and ten (50%) consultations were undertaken for adverse reactions post COVID-19 vaccination, with 84 (76%) concerning immediate symptoms. Thirty patients (27%) who had a consultation had had adrenaline administered post vaccination. One patient had biopsy confirmed Stevens Johnson Syndrome and was referred to Dermatology. All others due for further doses (107 patients) were recommended to have subsequent doses with 49 (45%) offered the same vaccine. Eighty-nine patients had a vaccine administered post adverse reaction and 79 (88%) tolerated the dose.
Skin testing and challenge to polyethylene glycol were negative in the 8 patients tested.
Over 1000 requests for advice and guidance were received during the review period, managed mainly with written advice. The overwhelming majority of requests for advice and consultations were for females, with equal distribution both pre- and post-COVID-19 vaccine administration. Vaccination was recommended in all but 1 patient (with biopsy confirmed Stevens Johnson Syndrome). Polyethylene glycol allergy was not confirmed in any patient, nor did any patient have confirmed anaphylaxis when the vaccine was administered under our supervision, suggesting that type 1 mediated hypersensitivity is uncommon even in this "high risk" population.
The SARS‐CoV‐2 virus causes COVID‐19, an infection capable of causing severe disease and death but which can also be asymptomatic or oligosymptomatic. We investigated whether ABO blood group or ...secretor status was associated with COVID‐19 severity. We investigated secretor status because expression of ABO glycans on secreted proteins and non‐erythroid cells are controlled by a fucosyltransferase (FUT2), and inactivating FUT2 mutations result in a non‐secretor phenotype which protects against some viral infections. Data combined from healthcare records and our own laboratory tests (n = 275) of hospitalized SARS‐CoV‐2 polymerase chain reaction positive patients confirmed higher than expected numbers of blood group A individuals compared to O (RR = 1.24, CI 95% 1.05, 1.47, p = 0.0111). There was also a significant association between group A and COVID‐19‐related cardiovascular complications (RR = 2.56, CI 95% 1.43, 4.55, p = 0.0011) which is independent of gender. Molecular analysis revealed that group A non‐secretors are significantly less likely to be hospitalized than secretors. Testing of convalescent plasma donors, among whom the majority displayed COVID‐19 symptoms and only a small minority required hospitalization, group A non‐secretors were slightly over‐represented. Our findings showed that group A non‐secretors are not resistant to infection by SARS‐CoV‐2, but are more likely to experience a less severe form of associated disease.
A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We ...evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials.
This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674.
Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0–75·7; 27 0·6% of 4440 in the ChAdOx1 nCoV-19 group vs71 1·6% of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4–97·0; three 0·2% of 1367 vs 30 2·2% of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8–80·6; 30 0·5% of 5807 vs 101 1·7% of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3–4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation.
ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials.
UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D’Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca.
The ChAdOx1 nCoV-19 (AZD1222) vaccine has been approved for emergency use by the UK regulatory authority, Medicines and Healthcare products Regulatory Agency, with a regimen of two standard doses ...given with an interval of 4–12 weeks. The planned roll-out in the UK will involve vaccinating people in high-risk categories with their first dose immediately, and delivering the second dose 12 weeks later. Here, we provide both a further prespecified pooled analysis of trials of ChAdOx1 nCoV-19 and exploratory analyses of the impact on immunogenicity and efficacy of extending the interval between priming and booster doses. In addition, we show the immunogenicity and protection afforded by the first dose, before a booster dose has been offered.
We present data from three single-blind randomised controlled trials—one phase 1/2 study in the UK (COV001), one phase 2/3 study in the UK (COV002), and a phase 3 study in Brazil (COV003)—and one double-blind phase 1/2 study in South Africa (COV005). As previously described, individuals 18 years and older were randomly assigned 1:1 to receive two standard doses of ChAdOx1 nCoV-19 (5 × 1010 viral particles) or a control vaccine or saline placebo. In the UK trial, a subset of participants received a lower dose (2·2 × 1010 viral particles) of the ChAdOx1 nCoV-19 for the first dose. The primary outcome was virologically confirmed symptomatic COVID-19 disease, defined as a nucleic acid amplification test (NAAT)-positive swab combined with at least one qualifying symptom (fever ≥37·8°C, cough, shortness of breath, or anosmia or ageusia) more than 14 days after the second dose. Secondary efficacy analyses included cases occuring at least 22 days after the first dose. Antibody responses measured by immunoassay and by pseudovirus neutralisation were exploratory outcomes. All cases of COVID-19 with a NAAT-positive swab were adjudicated for inclusion in the analysis by a masked independent endpoint review committee. The primary analysis included all participants who were SARS-CoV-2 N protein seronegative at baseline, had had at least 14 days of follow-up after the second dose, and had no evidence of previous SARS-CoV-2 infection from NAAT swabs. Safety was assessed in all participants who received at least one dose. The four trials are registered at ISRCTN89951424 (COV003) and ClinicalTrials.gov, NCT04324606 (COV001), NCT04400838 (COV002), and NCT04444674 (COV005).
Between April 23 and Dec 6, 2020, 24 422 participants were recruited and vaccinated across the four studies, of whom 17 178 were included in the primary analysis (8597 receiving ChAdOx1 nCoV-19 and 8581 receiving control vaccine). The data cutoff for these analyses was Dec 7, 2020. 332 NAAT-positive infections met the primary endpoint of symptomatic infection more than 14 days after the second dose. Overall vaccine efficacy more than 14 days after the second dose was 66·7% (95% CI 57·4–74·0), with 84 (1·0%) cases in the 8597 participants in the ChAdOx1 nCoV-19 group and 248 (2·9%) in the 8581 participants in the control group. There were no hospital admissions for COVID-19 in the ChAdOx1 nCoV-19 group after the initial 21-day exclusion period, and 15 in the control group. 108 (0·9%) of 12 282 participants in the ChAdOx1 nCoV-19 group and 127 (1·1%) of 11 962 participants in the control group had serious adverse events. There were seven deaths considered unrelated to vaccination (two in the ChAdOx1 nCov-19 group and five in the control group), including one COVID-19-related death in one participant in the control group. Exploratory analyses showed that vaccine efficacy after a single standard dose of vaccine from day 22 to day 90 after vaccination was 76·0% (59·3–85·9). Our modelling analysis indicated that protection did not wane during this initial 3-month period. Similarly, antibody levels were maintained during this period with minimal waning by day 90 (geometric mean ratio GMR 0·66 95% CI 0·59–0·74). In the participants who received two standard doses, after the second dose, efficacy was higher in those with a longer prime-boost interval (vaccine efficacy 81·3% 95% CI 60·3–91·2 at ≥12 weeks) than in those with a short interval (vaccine efficacy 55·1% 33·0–69·9 at <6 weeks). These observations are supported by immunogenicity data that showed binding antibody responses more than two-fold higher after an interval of 12 or more weeks compared with an interval of less than 6 weeks in those who were aged 18–55 years (GMR 2·32 2·01–2·68).
The results of this primary analysis of two doses of ChAdOx1 nCoV-19 were consistent with those seen in the interim analysis of the trials and confirm that the vaccine is efficacious, with results varying by dose interval in exploratory analyses. A 3-month dose interval might have advantages over a programme with a short dose interval for roll-out of a pandemic vaccine to protect the largest number of individuals in the population as early as possible when supplies are scarce, while also improving protection after receiving a second dose.
UK Research and Innovation, National Institutes of Health Research (NIHR), The Coalition for Epidemic Preparedness Innovations, the Bill & Melinda Gates Foundation, the Lemann Foundation, Rede D’Or, the Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca.
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