Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria.

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/ß-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/ß immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/ß. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/ß-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/ß-dependent antiviral immunity.

Development of antibody-based assays for high throughput discovery and mechanistic study of antiviral agents against yellow fever virus.

Despite the availability of a highly effective yellow fever virus (YFV) vaccine, outbreaks of yellow fever frequently occur in Africa and South America with significant mortality, highlighting the pressing need for antiviral drugs to manage future outbreaks. To support the discovery and development of antiviral drugs against YFV, we characterized a panel of rabbit polyclonal antibodies against the three YFV structural proteins and five non-structural proteins and demonstrated these antibody reagents in conjunction with viral RNA metabolic labeling, double-stranded RNA staining and membrane floatation assays as powerful tools for investigating YFV polyprotein processing, replication complex formation, viral RNA synthesis and high throughput discovery of antiviral drugs. Specifically, the proteolytic processing of the viral polyprotein can be analyzed by Western blot assays. The predominant nuclear localization of NS5 protein as well as the relationship between intracellular viral non-structural protein distribution and foci of YFV RNA replication can be revealed by immunofluorescence staining and membrane flotation assays. Using an antibody against YFV NS4B protein as an example, in-cell western and high-content imaging assays have been developed for high throughput discovery of antiviral agents. A synergistic antiviral effect of an YFV NS4B-targeting antiviral agent BDAA and a NS5 RNA-dependent RNA polymerase inhibitor (Sofosbuvir) was also demonstrated with the high-content imaging assay. Apparently, the antibody-based assays established herein not only facilitate the discovery and development of antiviral agents against YFV, but also provide valuable tools to dissect the molecular mechanism by which the antiviral agents inhibit YFV replication.

IFNL3-adjuvanted HCV DNA vaccine reduces regulatory T cell frequency and increases virus-specific T cell responses.

BACKGROUND & AIMS: Although direct-acting antiviral (DAA) treatment results in a sustained virologic response (SVR) in most patients with chronic HCV infection, they are at risk of re-infection. Moreover, the immune system is not completely normalized even after SVR (e.g. increased regulatory T [Treg] cell frequency). We developed a DNA vaccine, GLS-6150, to prevent re-infection of patients with DAA-induced SVR and evaluated its safety and immunogenicity in individuals with chronic HCV infection. METHODS: GLS-6150 consists of plasmids encoding HCV non-structural proteins (NS3-NS5A) and adjuvant IFNL3. The vaccine was administered 4 times at 4-weekly intervals to 3 groups (1, 3, or 6 mg/vaccination; n = 6 per group), followed by a 6 mg boost at 24 weeks (n = 14). Peripheral blood T cell responses were evaluated by interferon (IFN)-γ enzyme-linked immunospot assays, intracellular cytokine staining, and major histocompatibility complex class-I (MHC-I) dextramer staining. Treg cell frequency was assessed by flow cytometry. RESULTS: Severe adverse events or vaccine discontinuation were not reported. The IFN-γ spot-forming cells specific to NS3-NS5A were increased by GLS-6150. Both CD4+ and CD8+ T cells produced multiple cytokines. However, the frequency and phenotype of HCV-specific MHC-I dextramer+CD8+ T cells were not changed. Interestingly, the frequency of Treg cells, particularly activated Treg cells, was decreased by GLS-6150, as expected from previous reports that IFNL3 adjuvants decrease Treg cell frequency. Ex vivo IFN-λ3 treatment reduced Treg frequency in pre-vaccination peripheral blood mononuclear cells. Finally, Treg cell frequency inversely correlated with HCV-specific, IFN-γ-producing T cell responses in the study participants. CONCLUSIONS: We demonstrate that GLS-6150 decreases Treg cell frequency and enhances HCV-specific T cell responses without significant side effects. A phase I clinical trial of GLS-6150 is currently underway in patients with DAA-induced SVR. CLINICAL TRIAL NUMBER: NCT02027116. LAY SUMMARY: Although direct-acting antivirals (DAAs) are successfully used for the treatment of chronic hepatitis C virus (HCV) infection, a prophylactic HCV vaccine needs to be developed, especially for patients who achieve a sustained virologic response. In the current study, we show that a DNA vaccine (GLS-6150) was safe and increased HCV-specific T cell responses. A clinical trial is underway to test this vaccine in patients with a sustained virologic response following DAA therapy.

Structure-based glycoengineering of interferon lambda 4 enhances its productivity and anti-viral potency.

Interferon lambda 4 (IFNλ4) has been recently known and studied for its role in hepatitis C virus (HCV) infection, but its clinical potential is significantly hampered due to its poor expression in vitro. Our study reports the successful production of IFNλ4 from a mammalian cell line through a glycoengineering and structure-based approach. We introduced de novo N-glycosylation of IFNλ4, guided by structural analysis, and produced IFNλ4 variants in Expi293F that displayed improved expression and potency. To preserve the structure and functionality of IFNλ4, the model structure of the IFNλ4 signaling complex was analyzed and the N-glycosylation candidate sites were selected. The receptor binding activity of engineered IFNλ4 variants and their receptor-mediated signaling pathway were similar to the E. coli version of IFNλ4 (eIFNλ4), while the antiviral activity and induction levels of interferon-stimulated gene (ISG) were all more robust in our variants. Our engineered IFNλ4 variants may be further developed for clinical applications and utilized in basic research to decipher the immunological roles of IFNλ4.

Dynamic Changes in Ex Vivo T-Cell Function After Viral Clearance in Chronic HCV Infection.

BACKGROUND: Direct-acting antiviral (DAA) agents can successfully treat chronic hepatitis C virus (HCV) infection. However, the ex vivo HCV-specific T-cell function following viral clearance remains unknown. METHODS: We investigated functional alterations and phenotypic changes in ex vivo HCV-specific CD8+ T cells with a longitudinal analysis of 41 patients with chronic HCV infection who were undergoing DAA treatment. RESULTS: A patient subset exhibited a significantly increased T-cell response (mainly CD8+ T cells) at week 4 of treatment. However, this increased T-cell response diminished in later weeks. Relative to pretreatment levels, the ex vivo HCV-specific CD8+ T-cell frequency decreased at 12 weeks after the end of treatment, along with a decreased antigen-experienced CD8+ T-cell population. DAA treatment increased the proliferative capacity of HCV-specific CD8+ T cells, but this change was not correlated with ex vivo function. Patients experiencing viral breakthrough or relapse exhibited defective restoration of T-cell function. CONCLUSION: Our present results indicated that DAA-mediated viral clearance only transiently restored ex vivo T-cell function, suggesting a need to enhance T-cell function in DAA-treated patients.

Immune Checkpoint Inhibitor-induced Reinvigoration of Tumor-infiltrating CD8+ T Cells is Determined by Their Differentiation Status in Glioblastoma.

PURPOSE: Immune checkpoint inhibitors (ICI) are used for the treatment of various cancers, but clinical trials of anti-programmed cell death protein 1 (PD-1) with patients with recurrent glioblastoma (GBM) have failed to show clinical benefits. In this study, we examined the differentiation status of CD8+ tumor-infiltrating lymphocytes (TIL) from patients with primary GBM and their reinvigoration by ICIs to understand the nature of T-cell exhaustion in GBM. EXPERIMENTAL DESIGN: We isolated TILs from 98 patients with newly diagnosed GBM and examined the expression of immune checkpoint receptors and T-cell transcription factors using flow cytometry. TILs were ex vivo stimulated with anti-CD3 in the presence of anti-PD-1 and/or anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) and their proliferation assessed. RESULTS: CD8+ TILs had significantly increased expression of immune checkpoint receptors, including PD-1 and CTLA-4, compared with peripheral blood CD8+ T cells. Among CD8+ TILs, PD-1+ cells exhibited more terminally differentiated phenotypes (i.e., EomeshiT-betlo) than PD-1- cells. These data were confirmed by analyzing NY-ESO-1157-specific CD8+ TILs. Evaluating the proliferation of CD8+ TILs after ex vivo stimulation with anti-CD3 and anti-PD-1, we found that proliferation inversely correlated with the percentage of EomeshiT-betlo cells among PD-1+CD8+ TILs. When anti-CTLA-4 was used in combination with anti-PD-1, an additional increase in CD8+ TIL proliferation was observed in patients with low percentages of EomeshiT-betlo CD8+ TILs, who responded well to anti-PD-1 in ex vivo assays, but not in patients with high percentages of EomeshiT-betlo CD8+ TILs, who did not respond to anti-PD-1. CONCLUSIONS: In primary GBM, the differentiation status of CD8+ TILs determines their reinvigoration ability upon ICI treatment.

Is Chronic Exposure to Low-Dose Organochlorine Pesticides a New Risk Factor of T-cell Immunosenescence?

Background: T-cell immunosenescence, a hallmark of an aging immune system, is potentially linked to the risk of developing cancer and other aging-related diseases. Chronic infection by cytomegalovirus (CMV) has been widely studied as a risk factor for T-cell immunosenescence, but the role of persistent chemicals has never been examined. As a typical example of persistent chemicals, we evaluated whether organochlorine pesticides (OCPs) are related to T-cell immunosenescence in the general population.Methods: Serum concentrations of ß-hexachlorocyclohexane, p,p'-DDT, p,p'-DDE, and trans-nonachlor were measured in 95 Korean adults ages 30 to 64 years. T-cell immunosenescence was assessed by the frequencies of CD8+CD57+, CD8+CD28-, CD4+CD57+, and CD4+CD28- T lymphocytes in 20 mL of fresh peripheral blood.Results: The senescence of CD8+ T lymphocytes was the most consistently associated with OCPs. For quartiles of measurements of OCPs, adjusted mean percentages of CD8+CD57+ and CD8+CD28- T lymphocytes in the CD8+ T lymphocyte population were 23.9, 27.6, 31.0, and 38.7 (P trend < 0.01) and 25.6, 27.3, 28.0, and 35.5 (P trend = 0.02), respectively. When we compared the strength of the associations among OCPs, CMV IgG titer, and age, OCPs showed the strongest association with markers of immunosenescence. Importantly, the association between OCPs and immunosenescence markers was more prominent among participants without known risk factors, such as a young age or low CMV immunoglobulin G titer.Conclusions: Chronic exposure to low-dose OCPs may be a new risk factor for T-cell immunosenescence.Impact: T-cell immunosenescence may be one possible mechanism linking low-dose OCPs and many chronic diseases. Cancer Epidemiol Biomarkers Prev; 27(10); 1159-67. ©2018 AACR.

Innate-like Cytotoxic Function of Bystander-Activated CD8+ T Cells Is Associated with Liver Injury in Acute Hepatitis A.

Acute hepatitis A (AHA) involves severe CD8+ T cell-mediated liver injury. Here we showed during AHA, CD8+ T cells specific to unrelated viruses became activated. Hepatitis A virus (HAV)-infected cells produced IL-15 that induced T cell receptor (TCR)-independent activation of memory CD8+ T cells. TCR-independent activation of non-HAV-specific CD8+ T cells were detected in patients, as indicated by NKG2D upregulation, a marker of TCR-independent T cell activation by IL-15. CD8+ T cells derived from AHA patients exerted innate-like cytotoxicity triggered by activating receptors NKG2D and NKp30 without TCR engagement. We demonstrated that the severity of liver injury in AHA patients correlated with the activation of HAV-unrelated virus-specific CD8+ T cells and the innate-like cytolytic activity of CD8+ T cells, but not the activation of HAV-specific T cells. Thus, host injury in AHA is associated with innate-like cytotoxicity of bystander-activated CD8+ T cells, a result with implications for acute viral diseases.

Predictors of mortality in Middle East respiratory syndrome (MERS).

We evaluated the clinical characteristics, cytokine/chemokine concentrations, viral shedding and antibody kinetics in 30 patients with Middle East respiratory syndrome (MERS), including 6 non-survivors admitted to 3 MERS-designated hospitals. Old age, low albumin, altered mentality and high pneumonia severity index score at admission were risk factors for mortality. In addition, severe signs of inflammation at initial presentation (at hospital days 1-4), such as high inducible protein-10 (p=0.0013), monocyte chemoattractant protein-1 (p=0.0007) and interleukin 6 (p=0.0007) concentrations, and poor viral control (high viral load at hospital days 5-10, p<0.001) without adequate antibody titres (low antibody titre at hospital days 11-16, p=0.07) during the course of disease, were associated with mortality.

CXCL10 is produced in hepatitis A virus-infected cells in an IRF3-dependent but IFN-independent manner.

Acute hepatitis A caused by hepatitis A virus (HAV) infection is accompanied by severe liver injury in adult patients, and the liver injury is associated with the production of chemokines. Herein, we investigated the mechanism of how HAV infection induces the production of CXCR3 and CCR5 chemokines, such as CXCL10, CCL4 and CCL5. The production of CXCL10, CCL4 and CCL5 was markedly increased by HAV (HM-175/18f) infection in the culture of primary human hepatocytes and HepG2 cells. In particular, CXCL10 was produced in HAV-infected cells, not in neighboring uninfected cells. Moreover, these chemokines were significantly increased in the sera of acute hepatitis A patients. The production of IFN-λs was also robustly induced by HAV infection, and the blocking of secreted IFN-λs partially abrogated the production of CCL4 and CCL5 in HAV-infected cells. However, CXCL10 production was not decreased by the blocking of IFN-λs. Instead, CXCL10 production was reduced by silencing the expression of RIG-I-like receptor (RLR) signal molecules, such as mitochondrial antiviral signaling protein and interferon regulatory factor 3, in HAV-infected cells. In conclusion, HAV infection strongly induces the production of helper 1 T cell-associated chemokines, particularly CXCL10 via RLR signaling, even without secreted IFNs.

IFN-λ4 potently blocks IFN-α signalling by ISG15 and USP18 in hepatitis C virus infection.

Genetic polymorphisms in IFNL4 have been shown to predict responses to IFN-α-based therapy in hepatitis C virus (HCV)-infected patients. The IFNL4-ΔG genotype, which encodes functional IFN-λ4 protein, is associated with a poor treatment response. In the present study, we investigated the induction and biological effects of IFN-λ4 in HCV-infected hepatocytes and their association with responsiveness to IFN-α. We also studied the effects of direct-acting antiviral (DAA) treatment on IFN-λ4 expression and IFN-α responsiveness. HCV infection induced IFN-λ4 expression at mRNA and protein levels in primary human hepatocytes (PHHs). In hepatoma cells, IFNL4 gene transfection or recombinant IFN-λ4 protein treatment robustly increased the protein levels of ISG15 and USP18 in an IFNLR1-dependent manner and potently blocked IFN-α signalling. The ISG15/USP18-mediated IFN-α unresponsiveness was demonstrated by transfection of siRNAs targeting ISG15 and/or USP18. This potent IFN-λ4 effect was related to prolonged ISG expression after IFNL4 gene transfection. DAA treatment of HCV-infected PHHs reduced the expression of IFN-λs, including IFN-λ4, and restored IFN-α responsiveness. These results demonstrate that virus-induced IFN-λ4 potently blocks IFN-α signalling by inducing high protein levels of ISG15 and USP18. Moreover, the data clearly demonstrate that DAA therapy restores IFN-α responsiveness in HCV-infected cells.

Complete prevention of blood loss with self-sealing haemostatic needles.

Bleeding is largely unavoidable following syringe needle puncture of biological tissues and, while inconvenient, this typically causes little or no harm in healthy individuals. However, there are certain circumstances where syringe injections can have more significant side effects, such as uncontrolled bleeding in those with haemophilia, coagulopathy, or the transmission of infectious diseases through contaminated blood. Herein, we present a haemostatic hypodermic needle able to prevent bleeding following tissue puncture. The surface of the needle is coated with partially crosslinked catechol-functionalized chitosan that undergoes a solid-to-gel phase transition in situ to seal punctured tissues. Testing the capabilities of these haemostatic needles, we report complete prevention of blood loss following intravenous and intramuscular injections in animal models, and 100% survival in haemophiliac mice following syringe puncture of the jugular vein. Such self-sealing haemostatic needles and adhesive coatings may therefore help to prevent complications associated with bleeding in more clinical settings.

Immunoproteasome induction is suppressed in hepatitis C virus-infected cells in a protein kinase R-dependent manner.

By changing the relative abundance of generated antigenic peptides through alterations in the proteolytic activity, interferon (IFN)-γ-induced immunoproteasomes influence the outcome of CD8+ cytotoxic T lymphocyte responses. In the present study, we investigated the effects of hepatitis C virus (HCV) infection on IFN-γ-induced immunoproteasome expression using a HCV infection cell culture system. We found that, although IFN-γ induced the transcriptional expression of mRNAs encoding the ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 immunoproteasome subunits, the formation of immunoproteasomes was significantly suppressed in HCV-infected cells. This finding indicated that immunoproteasome induction was impaired at the translational or posttranslational level by HCV infection. Gene silencing studies showed that the suppression of immunoproteasome induction is essentially dependent on protein kinase R (PKR). Indeed, the generation of a strictly immunoproteasome-dependent cytotoxic T lymphocyte epitope was impaired in in vitro processing experiments using isolated 20S proteasomes from HCV-infected cells and was restored by the silencing of PKR expression. In conclusion, our data point to a novel mechanism of immune regulation by HCV that affects the antigen-processing machinery through the PKR-mediated suppression of immunoproteasome induction in infected cells.

Roles of unphosphorylated ISGF3 in HCV infection and interferon responsiveness.

Up-regulation of IFN-stimulated genes (ISGs) is sustained in hepatitis C virus (HCV)-infected livers. Here, we investigated the mechanism of prolonged ISG expression and its role in IFN responsiveness during HCV infection in relation to unphosphorylated IFN-stimulated gene factor 3 (U-ISGF3), recently identified as a tripartite transcription factor formed by high levels of IFN response factor 9 (IRF9), STAT1, and STAT2 without tyrosine phosphorylation of the STATs. The level of U-ISGF3, but not tyrosine phosphorylated STAT1, is significantly elevated in response to IFN-λ and IFN-ß during chronic HCV infection. U-ISGF3 prolongs the expression of a subset of ISGs and restricts HCV chronic replication. However, paradoxically, high levels of U-ISGF3 also confer unresponsiveness to IFN-α therapy. As a mechanism of U-ISGF3-induced resistance to IFN-α, we found that ISG15, a U-ISGF3-induced protein, sustains the abundance of ubiquitin-specific protease 18 (USP18), a negative regulator of IFN signaling. Our data demonstrate that U-ISGF3 induced by IFN-λs and -ß drives prolonged expression of a set of ISGs, leading to chronic activation of innate responses and conferring a lack of response to IFN-α in HCV-infected liver.