5,6-dimethylxanthenone-4-acetic acid (DMXAA), a partial STING agonist, competes for human STING activation.

5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a mouse-selective stimulator of interferon gene (STING) agonist exerting STING-dependent anti-tumor activity. Although DMXAA cannot fully activate human STING, DMXAA reached phase III in lung cancer clinical trials. How DMXAA is effective against human lung cancer is completely unknown. Here, we show that DMXAA is a partial STING agonist interfering with agonistic STING activation, which may explain its partial anti-tumor effect observed in humans, as STING was reported to be pro-tumorigenic for lung cancer cells with low antigenicity. Furthermore, we developed a DMXAA derivative-3-hydroxy-5-(4-hydroxybenzyl)-4-methyl-9H-xanthen-9-one (HHMX)-that can potently antagonize STING-mediated immune responses both in humans and mice. Notably, HHMX suppressed aberrant responses induced by STING gain-of-function mutations causing STING-associated vasculopathy with onset in infancy (SAVI) in in vitro experiments. Furthermore, HHMX treatment suppressed aberrant STING pathway activity in peripheral blood mononuclear cells from SAVI patients. Lastly, HHMX showed a potent therapeutic effect in SAVI mouse model by mitigating disease progression. Thus, HHMX offers therapeutic potential for STING-associated autoinflammatory diseases.

Immunogenicity and Protective Capacity of CpG ODN Adjuvanted Alum Adsorbed Bivalent Meningococcal Outer Membrane Vesicle Vaccine.

Invasive meningococcal disease (IMD) is caused by Neisseria meningitidis, with the main serogroups responsible for the disease being A, B, C, W, X, and Y. To date, several vaccines targeting N.meningitidis have been developed albeit with a short-lived protection. Given that MenW and MenB are the most common causes of IMD in Europe, Turkey, and Middle East, we aimed to develop an outer membrane vesicle (OMV) based bivalent vaccine as the heterologous antigen source. Herein, we compared the immunogenicity, and breadth of serum bactericidal assays (SBA) based protective coverage of OMV vaccine to X serotype with existing commercial meningococcal conjugate and polysaccharide (PS) vaccines in a murine model. BALB/c mice were immunized with preclinical batches of the W+B OMV vaccine, either adjuvanted with Alum, CpG ODN or their combinations and compared with a MenACYW conjugate vaccine (NimenrixTM, Pfizer) and a MenB OMV-based vaccine (Bexsero®, GSK), The immune responses were assessed through ELISA and SBA. Antibody responses and SBA titers were significantly higher in the W+B OMV vaccine when adjuvanted with Alum or CpG ODN, as compared to the control groups. Moreover, the SBA titers were not only significantly higher than those achieved with available conjugated ACYW vaccines but also on par with the 4CMenB vaccines. In conclusion, the W+B OMV vaccine demonstrated the capacity to elicit robust antibody responses, surpassing or matching the levels induced by licensed meningococcal vaccines. Consequently, the W+B OMV vaccine could potentially serve as a viable alternative or supplement to existing meningococcal vaccines.

DOCK8 deficiency with hypereosinophilia and the syndrome of inappropriate antidiuretic hormone secretion during herpes infection.

BACKGROUND: Hyperimmunoglobulin E syndrome (HIES) due to dedicator of cytokinesis8 (DOCK8) deficiency may present in infancy and childhood with different clinical features involving recurrent infections, allergic dysregulation, and autoimmunity. CASE: In this report, we describe a patient who first presented with severe hypereosinophilia and went on to develop the syndrome of inappropriate antidiuretic hormone secretion (SIADH) in the context of a severe herpes infection. Investigation revealed the presence of underlying DOCK8 deficiency presenting with atypical clinical features. CONCLUSIONS: Distinct inflammatory features associated with infections may be seen in the course of primary immunodeficiency diseases, and early functional and molecular genetic tests will aid the proper management.

Leishmania kinetoplast DNA contributes to parasite burden in infected macrophages: Critical role of the cGAS-STING-TBK1 signaling pathway in macrophage parasitemia.

Leishmania parasites harbor a unique network of circular DNA known as kinetoplast DNA (kDNA). The role of kDNA in leishmania infections is poorly understood. Herein, we show that kDNA delivery to the cytosol of Leishmania major infected THP-1 macrophages provoked increased parasite loads when compared to untreated cells, hinting at the involvement of cytosolic DNA sensors in facilitating parasite evasion from the immune system. Parasite proliferation was significantly hindered in cGAS- STING- and TBK-1 knockout THP-1 macrophages when compared to wild type cells. Nanostring nCounter gene expression analysis on L. major infected wild type versus knockout cells revealed that some of the most upregulated genes including, Granulysin (GNLY), Chitotriosidase-1 (CHIT1), Sialomucin core protein 24 (CD164), SLAM Family Member 7 (SLAMF7), insulin-like growth factor receptor 2 (IGF2R) and apolipoprotein E (APOE) were identical in infected cGAS and TBK1 knockout cells, implying their involvement in parasite control. Amlexanox treatment (a TBK1 inhibitor) of L. major infected wild type cells inhibited both the percentage and the parasite load of infected THP-1 cells and delayed footpad swelling in parasite infected mice. Collectively, these results suggest that leishmania parasites might hijack the cGAS-STING-TBK1 signaling pathway to their own advantage and the TBK1 inhibitor amlexanox could be of interest as a candidate drug in treatment of cutaneous leishmaniasis.

Low Density Granulocytes and Dysregulated Neutrophils Driving Autoinflammatory Manifestations in NEMO Deficiency.

NF-κB essential modulator (NEMO, IKK-γ) deficiency is a rare combined immunodeficiency caused by mutations in the IKBKG gene. Conventionally, patients are afflicted with life threatening recurrent microbial infections. Paradoxically, the spectrum of clinical manifestations includes severe inflammatory disorders. The mechanisms leading to autoinflammation in NEMO deficiency are currently unknown. Herein, we sought to investigate the underlying mechanisms of clinical autoinflammatory manifestations in a 12-years old male NEMO deficiency (EDA-ID, OMIM #300,291) patient by comparing the immune profile of the patient before and after hematopoietic stem cell transplantation (HSCT). Response to NF-kB activators were measured by cytokine ELISA. Neutrophil and low-density granulocyte (LDG) populations were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMC) transcriptome before and after HSCT and transcriptome of sorted normal-density neutrophils and LDGs were determined using the NanoString nCounter gene expression panels. ISG15 expression and protein ISGylation was based on Immunoblotting. Consistent with the immune deficiency, PBMCs of the patient were unresponsive to toll-like and T cell receptor-activators. Paradoxically, LDGs comprised 35% of patient PBMCs and elevated expression of genes such as MMP9, LTF, and LCN2 in the granulocytic lineage, high levels of IP-10 in the patient's plasma, spontaneous ISG15 expression and protein ISGylation indicative of a spontaneous type I interferon (IFN) signature were observed, all of which normalized after HSCT. Collectively, our results suggest that type I IFN signature observed in the patient, dysregulated LDGs and spontaneously activated neutrophils, potentially contribute to tissue damage in NEMO deficiency.

Expanding the Clinical and Immunological Phenotypes and Natural History of MALT1 Deficiency.

PURPOSE: MALT1 deficiency is a combined immune deficiency characterized by recurrent infections, eczema, chronic diarrhea, and failure to thrive. Clinical and immunological characterizations of the disease have not been previously reported in large cohorts. We sought to determine the clinical, immunological, genetic features, and the natural history of MALT-1 deficiency. METHODS: The clinical findings and treatment outcomes were evaluated in nine new MALT1-deficient patients. Peripheral lymphocyte subset analyses, cytokine secretion, and proliferation assays were performed. We also analyzed ten previously reported patients to comprehensively evaluate genotype/phenotype correlation. RESULTS: The mean age of patients and disease onset were 33 ± 17 and 1.6 ± 0.7 months, respectively. The main clinical findings of the disease were recurrent infections (100%), skin involvement (100%), failure to thrive (100%), oral lesions (67%), chronic diarrhea (56%), and autoimmunity (44%). Eosinophilia and high IgE were observed in six (67%) and two (22%) patients, respectively. The majority of patients had normal T and NK cells, while eight (89%) exhibited reduced B cells. Immunoglobulin replacement and antibiotics prophylaxis were mostly ineffective in reducing the frequency of infections and other complications. One patient received hematopoietic stem cell transplantation (HSCT) and five patients died as a complication of life-threatening infections. Analyzing this cohort with reported patients revealed overall survival in 58% (11/19), which was higher in patients who underwent HSCT (P = 0.03). CONCLUSION: This cohort provides the largest analysis for clinical and immunological features of MALT1 deficiency. HSCT should be offered as a curative therapeutic option for all patients at the early stage of life.

Development and preclinical evaluation of virus-like particle vaccine against COVID-19 infection.

BACKGROUND: Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the four structural proteins of SARS-CoV-2. METHODS: VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable-resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. RESULTS: Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN-adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1-biased T-cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals. CONCLUSION: These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.

Achieving global equity for COVID-19 vaccines: Stronger international partnerships and greater advocacy and solidarity are needed.

Peter Figueroa and co-authors advocate for equity in the worldwide provision of COVID-19 vaccines.

Global public health security and justice for vaccines and therapeutics in the COVID-19 pandemic.

A Lancet Commission for COVID-19 task force is shaping recommendations to achieve vaccine and therapeutics access, justice, and equity. This includes ensuring safety and effectiveness harmonized through robust systems of global pharmacovigilance and surveillance. Global production requires expanding support for development, manufacture, testing, and distribution of vaccines and therapeutics to low- and middle-income countries (LMICs). Global intellectual property rules must not stand in the way of research, production, technology transfer, or equitable access to essential health tools, and in context of pandemics to achieve increased manufacturing without discouraging innovation. Global governance around product quality requires channelling widely distributed vaccines through WHO prequalification (PQ)/emergency use listing (EUL) mechanisms and greater use of national regulatory authorities. A World Health Assembly (WHA) resolution would facilitate improvements and consistency in quality control and assurances. Global health systems require implementing steps to strengthen national systems for controlling COVID-19 and for influenza vaccinations for adults including pregnant and lactating women. A collaborative research network should strive to establish open access databases for bioinformatic analyses, together with programs directed at human capacity utilization and strengthening. Combating anti-science recognizes the urgency for countermeasures to address a global-wide disinformation movement dominating the internet and infiltrating parliaments and local governments.

Stepwise Reversal of Immune Dysregulation Due to STAT1 Gain-of-Function Mutation Following Ruxolitinib Bridge Therapy and Transplantation.

PURPOSE: Patients with heterozygous gain-of-function (GOF) mutations in STAT1 frequently exhibit chronic mucocutaneous candidiasis (CMC), immunodeficiency and autoimmune manifestations. Several treatment options including targeted therapies and hematopoietic stem cell transplantation (HSCT) are available for STAT1 GOF patients but modalities and outcomes are not well established. Herein, we aimed to unravel the effect of ruxolitinib as a bridge therapy in a patient with sporadic STAT1 T385M mutation to manage infections and other disease manifestations. METHODS: Peripheral blood mononuclear cells were isolated from the patient prior to, during ruxolitinib treatment and 6 months after HSCT. IFN-ß-induced STAT1 phosphorylation/dephosphorylation levels and PMA/ionomycin-stimulated intracellular IL-17A/IFN-γ production in CD4+ T cells were evaluated. Differentially expressed genes between healthy controls and the patient prior to, during ruxolitinib treatment and post-transplantation were investigated using Nanostring nCounter Profiling Panel. RESULTS: Ruxolitinib provided favorable responses by controlling candidiasis and autoimmune hemolytic anemia in the patient. Dysregulation in STAT1 phosphorylation kinetics improved with ruxolitinib treatment and was completely normalized after transplantation. TH17 deficiency persisted after ruxolitinib treatment, but normalized following HSCT. Consistent with the impairment in JAK/STAT signaling, multiple immune related pathways were found to be dysregulated in the patient. At baseline, genes related to type I IFN-related pathways, antigen processing, T-cell and B-cell functions were upregulated, while NK-cell function and cytotoxicity related genes were downregulated. Dysregulated gene expression was partially improved with ruxolitinib treatment and normalized after transplantation. CONCLUSION: Our findings suggest that improved disease management and immune dysregulatory profile can be achieved with ruxolitinib treatment before transplantation and this would be beneficial to reduce the risk of adverse outcome of HSCT.

Human Gut Commensal Membrane Vesicles Modulate Inflammation by Generating M2-like Macrophages and Myeloid-Derived Suppressor Cells.

Immunomodulatory commensal bacteria modify host immunity through delivery of regulatory microbial-derived products to host cells. Extracellular membrane vesicles (MVs) secreted from symbiont commensals represent one such transport mechanism. How MVs exert their anti-inflammatory effects or whether their tolerance-inducing potential can be used for therapeutic purposes remains poorly defined. In this study, we show that MVs isolated from the human lactic acid commensal bacteria Pediococcus pentosaceus suppressed Ag-specific humoral and cellular responses. MV treatment of bone marrow-derived macrophages and bone marrow progenitors promoted M2-like macrophage polarization and myeloid-derived suppressor cell differentiation, respectively, most likely in a TLR2-dependent manner. Consistent with their immunomodulatory activity, MV-differentiated cells upregulated expression of IL-10, arginase-1, and PD-L1 and suppressed the proliferation of activated T cells. MVs' anti-inflammatory effects were further tested in acute inflammation models in mice. In carbon tetrachloride-induced fibrosis and zymosan-induced peritonitis models, MVs ameliorated inflammation. In the dextran sodium sulfate-induced acute colitis model, systemic treatment with MVs prevented colon shortening and loss of crypt architecture. In an excisional wound healing model, i.p. MV administration accelerated wound closure through recruitment of PD-L1-expressing myeloid cells to the wound site. Collectively, these results indicate that P. pentosaceus-derived MVs hold promise as therapeutic agents in management/treatment of inflammatory conditions.

Dual-adjuvant effect of pH-sensitive liposomes loaded with STING and TLR9 agonists regress tumor development by enhancing Th1 immune response.

Nucleic acid-based pattern recognition receptor agonists are effective adjuvants and immunotherapeutic agents. Rather than single applications, ligand combinations could synergistically potentiate immune responses by elevating cytokine and chemokine production via triggering multiple signaling pathways. However, short half-lives of such labile ligands due to nuclease attack and limited cellular uptake due to their structure significantly hamper their in vivo performances. More importantly, simultaneous delivery and activity presentation of protein antigen and nucleic acid ligands critically limit the clinical development of these constructs. In this work, we approached this problem by co-encapsulating a model antigen ovalbumin along with TLR9 and STING ligands within liposomes, a well-established drug delivery system that enables payload stability and enhanced cellular activity upon internalization. Moreover, by loading dual ligands we postulated to achieve heightened Th-1 immune response that would yield pronounced protective vaccine efficacy. We show that, pH-sensitive liposomes co-encapsulating CpG ODN and cGAMP induced synergistic innate immune response by elevating type I and type II interferon levels. Most importantly, this vaccine formulation led to ~70% regression of established melanoma tumor. pH-sensitive liposomal vaccine administration elevated IgG2c/IgG1 antibody ratio, indicative of augmented OVA-specific Th1-biased immunity. Importantly, while the frequency of tumor-specific IFN-γ producing CD8+ T-cells was significantly increased, the M2-type anti-inflammatory macrophage levels were decreased in the tumor bed. In conclusion, our strategy induces reversal of immunosuppressive tumor microenvironment, while enhancing effective anti-tumor immune-response. We propose that this could be coupled with standard therapies during combating tumor eradication.

Autoinflammation in addition to combined immunodeficiency: SLC29A3 gene defect.

INTRODUCTION: H Syndrome is an autosomal recessive (AR) disease caused by defects in SLCA29A3 gene. This gene encodes the equilibrative nucleoside transporter, the protein which is highly expressed in spleen, lymph node and bone marrow. Autoinflammation and autoimmunity accompanies H Syndrome (HS). AIM: The aim was to further elucidate the mechanisms of disease by molecular studies in a patient with SLC29A3 gene defect. PATIENT AND METHODS: Mitochondrial dysfunction, lysosomal integrity, cytokine response in response to stimulation with different pattern recognition receptor ligands, and circulating cell-free mitochondrial-DNA(ccf-mtDNA) level in plasma were analyzed compared to controls to understand the cellular triggers of autoinflammation. RNA sequencing (RS) analyses were also performed in monocytes before/after culture with lipopolysaccharide. RESULTS: Patient had progressive destructive arthropathy in addition to clinical findings due to combined immunodeficiency. Pure red cell aplasia (PRCA), vitiligo, diabetes, multiple autoantibody positivity, lymphopenia, increased acute phase reactants were present. Recent thymic emigrants (RTE), naïve T cells were decreased, effector memory CD4 + T cells, nonclassical inflammatory monocytes were increased. Patient's peripheral blood mononuclear cells secreted more IL-1ß and IL-6, showed lysosomal disruption and significant mitochondrial dysfunction compared to healthy controls. Plasma ccf-mtDNA level was significantly elevated compared to age-matched controls (p < 0.05). RNA sequencing studies revealed decreased expression of NLR Family Caspase Recrument-Domain Containing 4(NLRC4), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4(PFKFB4), serine dehydratase(SDS), heparan sulfate(Glucosamine) 3-O-sulfotransferase 1(HS3ST1), neutral cholesterol ester hydrolase 1 (NCEH1), and interleukin-8 (IL-8) in patient's monocytes compared to controls. Longstanding PRCA, which is possibly autoimmune, resolved after initiating monthly intravenous immunoglobulins (IVIG) and low dose steroids to the patient. CONCLUSION: Although autoinflammation and autoimmunity are reported in HS, by functional analyses we here show in the present patient that over-active inflammasome pathway in HS might be related with mitochondrial and lysosomal dysfunction. Increased plasma ccf-mtDNA may be used as a biomarker of inflammasomopathy in HS. HS should be included in the classification of primary immunodeficiency diseases.

A suppressive oligodeoxynucleotide expressing TTAGGG motifs modulates cellular energetics through the mTOR signaling pathway.

Immune-mediated inflammation must be down-regulated to facilitate tissue remodeling during homeostatic restoration of an inflammatory response. Uncontrolled or over-exuberant immune activation can cause autoimmune diseases, as well as tissue destruction. A151, the archetypal example of a chemically synthesized suppressive oligodeoxynucleotide (ODN) based on repetitive telomere-derived TTAGGG sequences, was shown to successfully down-regulate a variety of immune responses. However, the degree, duration and breadth of A151-induced transcriptome alterations remain elusive. Here, we performed a comprehensive microarray analysis in combination with Ingenuity Pathway Analysis (IPA) using murine splenocytes to investigate the underlying mechanism of A151-dependent immune suppression. Our results revealed that A151 significantly down-regulates critical mammalian target of rapamycin (mTOR) activators (Pi3kcd, Pdpk1 and Rheb), elements downstream of mTOR signaling (Rps6ka1, Myc, Stat3 and Slc2a1), an important component of the mTORC2 protein complex (Rictor) and Mtor itself. The effects of A151 on mTOR signaling were dose- and time-dependent. Moreover, flow cytometry and immunoblotting analyses demonstrated that A151 is able to reverse mTOR phosphorylation comparably to the well-known mTOR inhibitor rapamycin. Furthermore, Seahorse metabolic assays showed an A151 ODN-induced decrease in both oxygen consumption and glycolysis implying that a metabolically inert state in macrophages could be triggered by A151 treatment. Overall, our findings suggested novel insights into the mechanism by which the immune system is metabolically modulated by A151 ODN.

Type I IFN-related NETosis in ataxia telangiectasia and Artemis deficiency.

BACKGROUND: Pathological inflammatory syndromes of unknown etiology are commonly observed in ataxia telangiectasia (AT) and Artemis deficiency. Similar inflammatory manifestations also exist in patients with STING-associated vasculopathy in infancy (SAVI). OBJECTIVE: We sought to test the hypothesis that the inflammation-associated manifestations observed in patients with AT and Artemis deficiency stem from increased type I IFN signature leading to neutrophil-mediated pathological damage. METHODS: Cytokine/protein signatures were determined by ELISA, cytometric bead array, or quantitative PCR. Stat1 phosphorylation levels were determined by flow cytometry. DNA species accumulating in the cytosol of patients' cells were quantified microscopically and flow cytometrically. Propensity of isolated polymorhonuclear granulocytes to form neutrophil extracellular traps (NETs) was determined using fluorescence microscopy and picogreen assay. Neutrophil reactive oxygen species levels and mitochondrial stress were assayed using fluorogenic probes, microscopy, and flow cytometry. RESULTS: Type I and III IFN signatures were elevated in plasma and peripheral blood cells of patients with AT, Artemis deficiency, and SAVI. Chronic IFN production stemmed from the accumulation of DNA in the cytoplasm of AT and Artemis-deficient cells. Neutrophils isolated from patients spontaneously produced NETs and displayed indicators of oxidative and mitochondrial stress, supportive of their NETotic tendencies. A similar phenomenon was also observed in neutrophils from healthy controls exposed to patient plasma samples or exogeneous IFN-α. CONCLUSIONS: Type I IFN-mediated neutrophil activation and NET formation may contribute to inflammatory manifestations observed in patients with AT, Artemis deficiency, and SAVI. Thus, neutrophils represent a promising target to manage inflammatory syndromes in diseases with active type I IFN signature.