Prevention of CD8 T Cell Deletion during Chronic Viral Infection.

During chronic viral infections, CD8 T cells rapidly lose antiviral and immune-stimulatory functions in a sustained program termed exhaustion. In addition to this loss of function, CD8 T cells with the highest affinity for viral antigen can be physically deleted. Consequently, treatments designed to restore function to exhausted cells and control chronic viral replication are limited from the onset by the decreased breadth of the antiviral T cell response. Yet, it remains unclear why certain populations of CD8 T cells are deleted while others are preserved in an exhausted state. We report that CD8 T cell deletion during chronic viral infection can be prevented by therapeutically lowering viral replication early after infection. The initial resistance to deletion enabled long-term maintenance of antiviral cytolytic activity of the otherwise deleted high-affinity CD8 T cells. In combination with decreased virus titers, CD4 T cell help and prolonged interactions with costimulatory molecules B7-1/B7-2 were required to prevent CD8 T cell deletion. Thus, therapeutic strategies to decrease early virus replication could enhance virus-specific CD8 T cell diversity and function during chronic infection.

The Antiviral Effect of the Chemical Compounds Targeting DED/EDh Motifs of the Viral Proteins on Lymphocytic Choriomeningitis Virus and SARS-CoV-2.

Arenaviruses and coronaviruses include several human pathogenic viruses, such as Lassa virus, Lymphocytic choriomeningitis virus (LCMV), SARS-CoV, MERS-CoV, and SARS-CoV-2. Although these viruses belong to different virus families, they possess a common motif, the DED/EDh motif, known as an exonuclease (ExoN) motif. In this study, proof-of-concept studies, in which the DED/EDh motif in these viral proteins, NP for arenaviruses, and nsp14 for coronaviruses, could be a drug target, were performed. Docking simulation studies between two structurally different chemical compounds, ATA and PV6R, and the DED/EDh motifs in these viral proteins indicated that these compounds target DED/EDh motifs. The concentration which exhibited modest cell toxicity was used with these compounds to treat LCMV and SARS-CoV-2 infections in two different cell lines, A549 and Vero 76 cells. Both ATA and PV6R inhibited the post-entry step of LCMV and SARS-CoV-2 infection. These studies strongly suggest that DED/EDh motifs in these viral proteins could be a drug target to combat two distinct viral families, arenaviruses and coronaviruses.

DAPK1 (death associated protein kinase 1) mediates mTORC1 activation and antiviral activities in CD8+ T cells.

Mechanistic target of rapamycin complex 1 (mTORC1) regulates CD8+ T-cell differentiation and function. Despite the links between PI3K-AKT and mTORC1 activation in CD8+ T cells, the molecular mechanism underlying mTORC1 activation remains unclear. Here, we show that both the kinase activity and the death domain of DAPK1 are required for maximal mTOR activation and CD8+ T-cell function. We found that TCR-induced activation of calcineurin activates DAPK1, which subsequently interacts with TSC2 via its death domain and phosphorylates TSC2 to mediate mTORC1 activation. Furthermore, both the kinase domain and death domain of DAPK1 are required for CD8+ T-cell antiviral responses in an LCMV infection model. Together, our data reveal a novel mechanism of mTORC1 activation that mediates optimal CD8+ T-cell function and antiviral activity.

Identification of Interleukin1ß as an Amplifier of Interferon alpha-induced Antiviral Responses.

The induction of an interferon-mediated response is the first line of defense against pathogens such as viruses. Yet, the dynamics and extent of interferon alpha (IFNα)-induced antiviral genes vary remarkably and comprise three expression clusters: early, intermediate and late. By mathematical modeling based on time-resolved quantitative data, we identified mRNA stability as well as a negative regulatory loop as key mechanisms endogenously controlling the expression dynamics of IFNα-induced antiviral genes in hepatocytes. Guided by the mathematical model, we uncovered that this regulatory loop is mediated by the transcription factor IRF2 and showed that knock-down of IRF2 results in enhanced expression of early, intermediate and late IFNα-induced antiviral genes. Co-stimulation experiments with different pro-inflammatory cytokines revealed that this amplified expression dynamics of the early, intermediate and late IFNα-induced antiviral genes can also be achieved by co-application of IFNα and interleukin1 beta (IL1ß). Consistently, we found that IL1ß enhances IFNα-mediated repression of viral replication. Conversely, we observed that in IL1ß receptor knock-out mice replication of viruses sensitive to IFNα is increased. Thus, IL1ß is capable to potentiate IFNα-induced antiviral responses and could be exploited to improve antiviral therapies.

PD-L1 Checkpoint Inhibition Narrows the Antigen-Specific T Cell Receptor Repertoire in Chronic Lymphocytic Choriomeningitis Virus Infection.

Checkpoint inhibitors are effective in restoring exhausted CD8+ T cell responses in persistent viral infections or tumors. Several compounds are in clinical use for different malignancies, but trials in patients with chronic viral infections have also been conducted. In a mouse model of persistent lymphocytic choriomeningitis virus (LCMV) infection, it was shown that checkpoint inhibitor treatment increased T cell proliferation and functionality, but its influence on the antigen-specific T cell receptor (TCR) repertoire is unknown. NP396-specific CD8+ T cells dominate during acute LCMV infection and are predominantly exhausted during chronic infection. Next-generation sequencing of NP396-specific TCRs showed that exhaustion corresponds with a significantly reduced NP396-specific TCR repertoire diversity: Shannon indices of 4 in immunized mice to 2.6 in persistently infected mice. Anti-PD-L1 treatment during persistent LCMV infection restored NP396-specific T cell responses and reduced viral titers. Nevertheless, anti-PD-L1-treated mice showed an even more narrowed TCR repertoire, with reduced TCR diversity compared to that of persistently infected control mice (Shannon indices of 2.1 and 2.6, respectively). Interestingly, anti-PD-L1 treatment-induced narrowing of the TCR repertoire negatively correlates with functional and physical restoration of the antigen-specific T cell response. Further, we found that private, hyperexpanded TCR clonotypes dominated the T cell response after anti-PD-L1 treatment. Although being private, these top clonotypes from anti-PD-L1-treated mice revealed a more closely related CDR3 motif than those of top clonotypes from persistently infected control mice. In conclusion, although targeting the PD-1/PD-L1 pathway reinvigorates exhausted CD8+ T cells, it fails to restore T cell repertoire diversity.IMPORTANCE Checkpoint inhibitors are effective immunotherapeutics to restore cancer- and virus-induced exhausted CD8+ T cells, by enhancing the quality and survival of immune responses. Although checkpoint inhibitors are already used as therapy against various cancers, not much is known about their multifaceted impact on the exhausted CD8+ T cell receptor (TCR) repertoire. This report describes for the first time the evolvement of an exhausted antigen-specific CD8+ TCR repertoire under checkpoint inhibitor treatment. By using a well-established virus model, we were able to show major shifts toward oligoclonality of the CD8+ TCR repertoire response against a massively exhausted lymphocytic choriomeningitis virus (LCMV) epitope. While supporting viral control in the LCMV model, oligoclonality and more private of TCR repertoires may impact future pathogenic challenges and may promote viral escape. Our results may explain the ongoing problems of viral escapes, unpredictable autoimmunity, and heterogeneous responses appearing as adverse effects of checkpoint inhibitor treatments.

The ReFRAME library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors.

Several mammarenaviruses, chiefly Lassa virus (LASV) in Western Africa and Junín virus (JUNV) in the Argentine Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. Moreover, mounting evidence indicates that the worldwide-distributed mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The lack of licensed mammarenavirus vaccines and partial efficacy of current anti-mammarenavirus therapy limited to an off-label use of the nucleoside analog ribavirin underscore an unmet need for novel therapeutics to combat human pathogenic mammarenavirus infections. This task can be facilitated by the implementation of "drug repurposing" strategies to reduce the time and resources required to advance identified antiviral drug candidates into the clinic. We screened a drug repurposing library of 11,968 compounds (Repurposing, Focused Rescue and Accelerated Medchem [ReFRAME]) and identified several potent inhibitors of LCMV multiplication that had also strong anti-viral activity against LASV and JUNV. Our findings indicate that enzymes of the rate-limiting steps of pyrimidine and purine biosynthesis, the pro-viral MCL1 apoptosis regulator, BCL2 family member protein and the mitochondrial electron transport complex III, play critical roles in the completion of the mammarenavirus life cycle, suggesting they represent potential druggable targets to counter human pathogenic mammarenavirus infections.

Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade.

T cell dysfunction is a hallmark of many cancers, but the basis for T cell dysfunction and the mechanisms by which antibody blockade of the inhibitory receptor PD-1 (anti-PD-1) reinvigorates T cells are not fully understood. Here we show that such therapy acts on a specific subpopulation of exhausted CD8+ tumor-infiltrating lymphocytes (TILs). Dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of exhaustion that mirror those seen in chronic viral infection. Exhausted CD8+ TILs include a subpopulation of 'progenitor exhausted' cells that retain polyfunctionality, persist long term and differentiate into 'terminally exhausted' TILs. Consequently, progenitor exhausted CD8+ TILs are better able to control tumor growth than are terminally exhausted T cells. Progenitor exhausted TILs can respond to anti-PD-1 therapy, but terminally exhausted TILs cannot. Patients with melanoma who have a higher percentage of progenitor exhausted cells experience a longer duration of response to checkpoint-blockade therapy. Thus, approaches to expand the population of progenitor exhausted CD8+ T cells might be an important component of improving the response to checkpoint blockade.

Metal chelators for the inhibition of the lymphocytic choriomeningitis virus endonuclease domain.

Arenaviridae is a viral family whose members are associated with rodent-transmitted infections to humans responsible of severe diseases. The current lack of a vaccine and limited therapeutic options make the development of efficacious drugs of high priority. The cap-snatching mechanism of transcription of Arenavirus performed by the endonuclease domain of the L-protein is unique and essential, so we developed a drug design program targeting the endonuclease activity of the prototypic Lymphocytic ChorioMeningitis Virus. Since the endonuclease activity is metal ion dependent, we designed a library of compounds bearing chelating motifs (diketo acids, polyphenols, and N-hydroxyisoquinoline-1,3-diones) able to block the catalytic center through the chelation of the critical metal ions, resulting in a functional impairment. We pre-screened 59 compounds by Differential Scanning Fluorimetry. Then, we characterized the binding affinity by Microscale Thermophoresis and evaluated selected compounds in in vitro and in cellula assays. We found several potent binders and inhibitors of the endonuclease activity. This study validates the proof of concept that the endonuclease domain of Arenavirus can be used as a target for anti-arena-viral drug discovery and that both diketo acids and N-hydroxyisoquinoline-1,3-diones can be considered further as potential metal-chelating pharmacophores.

Type I interferon signaling attenuates regulatory T cell function in viral infection and in the tumor microenvironment.

Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute, chronic infectious diseases and tumor microenvironment remains unclear. We recently demonstrated that IFN-α/ß receptor (IFNAR) signaling promotes Treg function in autoimmunity. Here we dissected the functional role of IFNAR-signaling in Tregs using Treg-specific IFNAR deficient (IFNARfl/flxFoxp3YFP-Cre) mice in acute LCMV Armstrong, chronic Clone-13 viral infection, and in tumor models. In both viral infection and tumor models, IFNARfl/flxFoxp3YFP-Cre mice Tregs expressed enhanced Treg associated activation antigens. LCMV-specific CD8+ T cells and tumor infiltrating lymphocytes from IFNARfl/flxFoxp3YFP-Cre mice produced less antiviral and antitumor IFN-γ and TNF-α. In chronic viral model, the numbers of antiviral effector and memory CD8+ T cells were decreased in IFNARfl/flxFoxp3YFP-Cre mice and the effector CD4+ and CD8+ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNARfl/flxFoxp3YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs during chronic infection, and higher tumor burden than the WT controls. The enhanced activated phenotype was evident through transcriptome analysis of IFNARfl/flxFoxp3YFP-Cre mice Tregs during infection demonstrated differential expression of a unique gene signature characterized by elevated levels of genes involved in suppression and decreased levels of genes mediating apoptosis. Thus, IFN signaling in Tregs is beneficial to host resulting in a more effective antiviral response and augmented antitumor immunity.

Mining a Kröhnke Pyridine Library for Anti-Arenavirus Activity.

Several arenaviruses cause hemorrhagic fever (HF) disease in humans and represent important public health problems in their endemic regions. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus is a neglected human pathogen of clinical significance. There are no licensed arenavirus vaccines, and current antiarenavirus therapy is limited to an off-label use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel therapeutics to combat human pathogenic arenaviruses, a task that will be facilitated by the identification of compounds with antiarenaviral activity that could serve as probes to identify arenavirus-host interactions suitable for targeting, as well as lead compounds to develop future antiarenaviral drugs. Screening of a combinatorial library of Krönhke pyridines identified compound KP-146 [(5-(5-(2,3-dihydrobenzo[ b][1,4] dioxin-6-yl)-4'-methoxy-[1,1'-biphenyl]-3-yl)thiophene-2-carboxamide] as having strong anti-lymphocytic choriomeningitis virus (LCMV) activity in cultured cells. KP-146 did not inhibit LCMV cell entry but rather interfered with the activity of the LCMV ribonucleoprotein (vRNP) responsible for directing virus RNA replication and gene transcription, as well as with the budding process mediated by the LCMV matrix Z protein. LCMV variants with increased resistance to KP-146 did not emerge after serial passages in the presence of KP-146. Our findings support the consideration of Kröhnke pyridine scaffold as a valuable source to identify compounds that could serve as tools to dissect arenavirus-host interactions, as well as lead candidate structures to develop antiarenaviral drugs.

T regulatory cells are critical for the maintenance, anamnestic expansion and protection elicited by vaccine-induced CD8 T cells.

T regulatory (Treg) cells are critical for preventing autoimmunity and suppressing immune responses during cancer and chronic infection. However, the role of Treg cells in the generation of vaccine-induced immune memory remains ill-defined. Using the mouse model of lymphocytic choriomeningitis virus (LCMV) infection, we demonstrate that transient absence of Treg cells during effector to memory CD8 T-cell transition results in a permanent impairment in the maintenance, function and recall capacity of CD8 T cells. Memory CD8 T cells in mice that were transiently depleted of Treg cells exhibited defective up-regulation of memory markers with a significant decrease in polyfunctionality. However, Treg-depleted mice showed no significant change in CD4 T-cell responses, and antibody levels relative to control. Altogether, this study evaluates the role of Treg cells in the formation of immune memory and demonstrates an important role for Treg cells in promoting memory CD8 T-cell differentiation and vaccine-induced immune protection against intracellular pathogens.

Virus-Induced Type I Interferon Deteriorates Control of Systemic Pseudomonas Aeruginosa Infection.

BACKGROUND: Type I interferon (IFN-I) predisposes to bacterial superinfections, an important problem during viral infection or treatment with interferon-alpha (IFN-α). IFN-I-induced neutropenia is one reason for the impaired bacterial control; however there is evidence that more frequent bacterial infections during IFN-α-treatment occur independently of neutropenia. METHODS: We analyzed in a mouse model, whether Pseudomonas aeruginosa control is influenced by co-infection with the lymphocytic choriomeningitis virus (LCMV). Bacterial titers, numbers of neutrophils and the gene-expression of liver-lysozyme-2 were determined during a 24 hours systemic infection with P. aeruginosa in wild-type and Ifnar(-/-) mice under the influence of LCMV or poly(I:C). RESULTS: Virus-induced IFN-I impaired the control of Pseudomonas aeruginosa. This was associated with neutropenia and loss of lysozyme-2-expression in the liver, which had captured P. aeruginosa. A lower release of IFN-I by poly(I:C)-injection also impaired the bacterial control in the liver and reduced the expression of liver-lysozyme-2. Low concentration of IFN-I after infection with a virulent strain of P. aeruginosa alone impaired the bacterial control and reduced lysozyme-2-expression in the liver as well. CONCLUSION: We found that during systemic infection with P. aeruginosa Kupffer cells quickly controlled the bacteria in cooperation with neutrophils. Upon LCMV-infection this cooperation was disturbed.

Au-ACRAMTU-PEt3 Alters Redox Balance To Inhibit T Cell Proliferation and Function.

Although T cells play a critical role in protection from viruses, bacteria, and tumors, they also cause autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis. Unwanted T cell responses during organ transplant, graft-versus-host disease, and allergies are also major clinical problems. Although drugs are available to suppress unwanted immune responses, they have limited efficacy with serious side effects. Thus, new therapeutics limiting T cell activation, proliferation, and function can make an immediate clinical impact. To identify new suppressors of lymphocyte activation, proliferation, and function, we examined the immunosuppressive activity of gold(I) analogs of platinum-acridine antitumor agents. We found that the gold complex Au-ACRAMTU-PEt3 is a potent suppressor of murine and human T cell activation. Preincubation with Au-ACRAMTU-PEt3 suppresses the proliferation of CD4(+) and CD8(+) T cells at a similar concentration as pharmaceutical grade cyclosporine A. Au-ACRAMTU-PEt3 pretreatment decreases the production of IFN-γ, TNF-α, IL-2, and IL-17 by human and murine CD4(+) and CD8(+) T cells. When mice were treated with Au-ACRAMTU-PEt3 during viral infection, the expansion of virus-specific CD8(+) T cells was decreased 10-fold and viral load was elevated. Taken together, these results demonstrate that Au-ACRAMTU-PEt3 has potent immunosuppressive activity that could be used to suppress immune responses during transplantation and autoimmunity.

Antiviral Effect of Interferon Lambda Against Lymphocytic Choriomeningitis Virus.

Lambda interferons inhibit replication of many viruses, but their role in the inhibition of lymphocytic choriomeningitis virus (LCMV) infection remains unclear. In this study, we examined the antiviral effects of interferon (IFN)-λ2 and IFN-λ3 against LCMV in A549 cells. We found that IFN-λ2 is a more potent inhibitor of LCMV strain MX compared with IFN-λ3, whereas both cytokines have similar antiviral effects against an immunosuppressive variant of LCMV, clone-13. We also demonstrated that the antiviral activity of IFN-λ2 is more effective if it is delivered early rather than after establishment of a long-term infection, suggesting that virus replication is only partially responsive to the cytokine. In agreement with this observation, we showed that LCMV infection significantly reduces IFNLR1 mRNA expression in infected cells. In addition, LCMV infection, to some extent, compromises the signal transduction pathway of IFN-λ2. This implies that IFN receptors as well as their downstream signaling components could be selectively targeted either directly by LCMV proteins or indirectly by cellular factor(s) that are induced or activated by LCMV infection.

Immune memory-boosting dose of rapamycin impairs macrophage vesicle acidification and curtails glycolysis in effector CD8 cells, impairing defense against acute infections.

Direct mammalian target of rapamycin (Rapa) complex 1 inhibition by short-term low-dose Rapa treatment has recently been shown to improve CD8 T cell immunological memory. Whereas these studies focused on memory development, the impact of low-dose Rapa on the primary immune response, particularly as it relates to functional effector immunity, is far less clear. In this study, we investigated the impact of acute Rapa treatment on immune effector cell function during the primary immune response to several acute infections. We found that functional CD8 T cell and macrophage responses to both viral and intracellular bacterial pathogens were depressed in mice in vivo and in humans to phorbol ester and calcium ionophore stimulation in vitro in the face of low-dose Rapa treatment. Mechanistically, the CD8 defect was linked to impaired glycolytic switch in stimulated naive cells and the reduced formation of short-lived effector cells. Therefore, more than one cell type required for a protective effector immune response is impaired by Rapa in both mice and humans, at the dose shown to improve immune memory and extend lifespan. This urges caution with regard to the relative therapeutic costs and benefits of Rapa treatment as means to improve immune memory.

Inhibition of multiplication of the prototypic arenavirus LCMV by valproic acid.

Valproic acid (VPA), a short chain fatty acid commonly used for treatment of neurological disorders, has been shown to inhibit production of infectious progeny of different enveloped viruses including the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). In this study we have investigated the mechanisms by which VPA inhibits LCMV multiplication in cultured cells. VPA reduced production of infectious LCMV progeny and virus propagation without exerting a major blockage on either viral RNA or protein synthesis, but rather affecting the cell release and specific infectivity of LCMV progeny from infected cells. Our results would support the repurposing of VPA as a candidate antiviral drug to combat arenavirus infections.

PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells.

The inhibitory receptor programmed cell death 1 (PD-1) plays a major role in functional exhaustion of T cells during chronic infections and cancer, and recent clinical data suggest that blockade of the PD-1 pathway is an effective immunotherapy in treating certain cancers. Thus, it is important to define combinatorial approaches that increase the efficacy of PD-1 blockade. To address this issue, we examined the effect of IL-2 and PD-1 ligand 1 (PD-L1) blockade in the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We found that low-dose IL-2 administration alone enhanced CD8+ T cell responses in chronically infected mice. IL-2 treatment also decreased inhibitory receptor levels on virus-specific CD8+ T cells and increased expression of CD127 and CD44, resulting in a phenotype resembling that of memory T cells. Surprisingly, IL-2 therapy had only a minimal effect on reducing viral load. However, combining IL-2 treatment with blockade of the PD-1 inhibitory pathway had striking synergistic effects in enhancing virus-specific CD8+ T cell responses and decreasing viral load. Interestingly, this reduction in viral load occurred despite increased numbers of Tregs. These results suggest that combined IL-2 therapy and PD-L1 blockade merits consideration as a regimen for treating human chronic infections and cancer.

Reactive oxygen species delay control of lymphocytic choriomeningitis virus.

Cluster of differentiation (CD)8(+) T cells are like a double edged sword during chronic viral infections because they not only promote virus elimination but also induce virus-mediated immunopathology. Elevated levels of reactive oxygen species (ROS) have been reported during virus infections. However, the role of ROS in T-cell-mediated immunopathology remains unclear. Here we used the murine lymphocytic choriomeningitis virus to explore the role of ROS during the processes of virus elimination and induction of immunopathology. We found that virus infection led to elevated levels of ROS producing granulocytes and macrophages in virus-infected liver and spleen tissues that were triggered by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Lack of the regulatory subunit p47phox of the NADPH oxidase diminished ROS production in these cells. While CD8(+) T cells exhibited ROS production that was independent of NADPH oxidase expression, survival and T-cell function was elevated in p47phox-deficient (Ncf1(-/-)) mice. In the absence of p47phox, enhanced T-cell immunity promoted virus elimination and blunted corresponding immunopathology. In conclusion, we find that NADPH-mediated production of ROS critically impairs the immune response, impacting elimination of virus and outcome of liver cell damage.

Arenaviruses and lethal mutagenesis. Prospects for new ribavirin-based interventions.

Lymphocytic choriomeningitis virus (LCMV) has contributed to unveil some of the molecular mechanisms of lethal mutagenesis, or loss of virus infectivity due to increased mutation rates. Here we review these developments, and provide additional evidence that ribavirin displays a dual mutagenic and inhibitory activity on LCMV that can be relevant to treatment designs. Using 5-fluorouracil as mutagenic agent and ribavirin either as inhibitor or mutagen, we document an advantage of a sequential inhibitor-mutagen administration over the corresponding combination treatment to achieve a low LCMV load in cell culture. This advantage is accentuated in the concentration range in which ribavirin acts mainly as an inhibitor, rather than as mutagen. This observation reinforces previous theoretical and experimental studies in supporting a sequential inhibitor-mutagen administration as a possible antiviral design. Given recent progress in the development of new inhibitors of arenavirus replication, our results suggest new options of ribavirin-based anti-arenavirus treatments.