RESUMO
Lujo virus (LUJV), a highly pathogenic arenavirus, was first identified in 2008 in Zambia. To aid the identification of effective therapeutics for LUJV, we developed a recombinant reporter virus system, confirming reporter LUJV comparability with wild-type virus and its utility in high-throughput antiviral screening assays. Using this system, we evaluated compounds with known and unknown efficacy against related arenaviruses, with the aim of identifying LUJV-specific and potential new pan-arenavirus antivirals. We identified six compounds demonstrating robust anti-LUJV activity, including several compounds with previously reported activity against other arenaviruses. These data provide critical evidence for developing broad-spectrum antivirals against high-consequence arenaviruses.
Assuntos
Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Lujo virus/efeitos dos fármacos , Animais , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/virologia , Arenavirus/fisiologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Genoma Viral , Proteínas de Fluorescência Verde/genética , Humanos , Lujo virus/genética , Lujo virus/fisiologia , Testes de Sensibilidade Microbiana , Proteínas Recombinantes , Células Vero , Internalização do Vírus/efeitos dos fármacosRESUMO
Several arenaviruses, including Lassa and Lujo viruses in Africa and five New World arenavirus (NWA) species in the Americas, cause life-threatening viral hemorrhagic fevers. In the absence of licensed antiviral therapies, these viruses pose a significant public health risk. The envelope glycoprotein complex (GPC) mediates arenavirus entry through a pH-dependent fusion of the viral and host endosomal membranes. It thus is recognized as a viable target for small-molecule fusion inhibitors. Here, we report on the antiviral activity and pre-clinical development of the novel broad-spectrum arenavirus fusion inhibitors, ARN-75039 and ARN-75041. In Tacaribe virus (TCRV) pseudotyped and native virus assays, the ARN compounds were active in the low to sub-nanomolar range with selectivity indices exceeding 1000. Pharmacokinetic analysis of the orally administered compounds revealed an extended half-life in mice supporting once-daily dosing, and the compounds were well tolerated at the highest tested dose of 100 mg/kg. In a proof-of-concept prophylactic efficacy study, doses of 10 and 35 mg/kg of either compound dramatically improved survival outcome and potently inhibited TCRV replication in serum and various tissues. Additionally, in contrast to surviving mice that received ribavirin or placebo, animals treated with ARN-75039 or ARN-75041 were cured of TCRV infection. In a follow-up study with ARN-75039, impressive therapeutic efficacy was demonstrated under conditions where treatment was withheld until after the onset of disease. Taken together, the data strongly support the continued development of ARN-75039 as a candidate therapeutic for the treatment of severe arenaviral diseases.
Assuntos
Antivirais/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Arenavirus do Novo Mundo/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Administração Oral , Animais , Antivirais/farmacocinética , Chlorocebus aethiops , Masculino , Camundongos , Ribavirina/farmacologia , Bibliotecas de Moléculas Pequenas/farmacocinética , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
Neglected diseases caused by arenaviruses such as Lassa virus (LASV) and filoviruses like Ebola virus (EBOV) primarily afflict resource-limited countries, where antiviral drug development is often minimal. Previous studies have shown that many approved drugs developed for other clinical indications inhibit EBOV and LASV and that combinations of these drugs provide synergistic suppression of EBOV, often by blocking discrete steps in virus entry. We hypothesize that repurposing of combinations of orally administered approved drugs provides effective suppression of arenaviruses. In this report, we demonstrate that arbidol, an approved influenza antiviral previously shown to inhibit EBOV, LASV, and many other viruses, inhibits murine leukemia virus (MLV) reporter viruses pseudotyped with the fusion glycoproteins (GPs) of other arenaviruses (Junin virus [JUNV], lymphocytic choriomeningitis virus [LCMV], and Pichinde virus [PICV]). Arbidol and other approved drugs, including aripiprazole, amodiaquine, sertraline, and niclosamide, also inhibit infection of cells by infectious PICV, and arbidol, sertraline, and niclosamide inhibit infectious LASV. Combining arbidol with aripiprazole or sertraline results in the synergistic suppression of LASV and JUNV GP-bearing pseudoviruses. This proof-of-concept study shows that arenavirus infection in vitro can be synergistically inhibited by combinations of approved drugs. This approach may lead to a proactive strategy with which to prepare for and control known and new arenavirus outbreaks.
Assuntos
Antivirais/uso terapêutico , Infecções por Arenaviridae/tratamento farmacológico , Arenavirus/efeitos dos fármacos , Administração Oral , Animais , Infecções por Arenaviridae/virologia , Linhagem Celular , Chlorocebus aethiops , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Células HEK293 , Humanos , Camundongos , Estudo de Prova de Conceito , Células VeroRESUMO
The potent antiviral properties of 3-deazaneplanocin, 3-deaza-isoneplanocins (1) and recently discovered l-like carbocyclic nucleosides (2, 3 and 4) prompted us to pursue rationally conceived l-like 3-deazaneplanocin analogues. The synthesis of those analogues including l-like 3-deazaneplanocin (5), l-like 3-bromo-3-deazaneplanocin (6), and l-like 5'-fluoro-5'-deoxy-3-deazaneplanocin (7), was accomplished from a common intermediate, (-)-cyclopentenone (8). Antiviral analysis found 5 and 6 to display favorable activity against the Ebola virus, as expected for 3-deazaadenine carbocyclic nucleosides. Compound 5 also showed activity against arenaviruses, including Pinchinde and Tacaribe.
Assuntos
Adenosina/análogos & derivados , Antivirais/síntese química , Antivirais/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Arenaviridae/efeitos dos fármacos , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Adenosina/química , Infecções por Arenaviridae/virologia , Doença pelo Vírus Ebola/virologia , Humanos , Estrutura MolecularRESUMO
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.
Assuntos
Antivirais/farmacologia , Arenaviridae/efeitos dos fármacos , Bases de Dados de Produtos Farmacêuticos , Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos/métodos , Células A549 , Animais , Apoptose , Arenaviridae/fisiologia , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/virologia , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Células HEK293 , Humanos , Interferons/genética , Vírus Junin/efeitos dos fármacos , Vírus Lassa/efeitos dos fármacos , Vírus da Coriomeningite Linfocítica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Purinas/biossíntese , Pirimidinas/biossíntese , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Some studies show eliminating senescent cells rejuvenate aged mice and attenuate deleterious effects of chemotherapy. Nevertheless, it remains unclear whether senescence affects immune cell function. We provide evidence that exposure of mice to ionizing radiation (IR) promotes the senescent-associated secretory phenotype (SASP) and expression of p16INK4a in splenic cell populations. We observe splenic T cells exhibit a reduced proliferative response when cultured with allogenic cells in vitro and following viral infection in vivo. Using p16-3MR mice that allow elimination of p16INK4a -positive cells with exposure to ganciclovir, we show that impaired T-cell proliferation is partially reversed, mechanistically dependent on p16INK4a expression and the SASP. Moreover, we found macrophages isolated from irradiated spleens to have a reduced phagocytosis activity in vitro, a defect also restored by the elimination of p16INK4a expression. Our results provide molecular insight on how senescence-inducing IR promotes loss of immune cell fitness, which suggest senolytic drugs may improve immune cell function in aged and patients undergoing cancer treatment.
Assuntos
Senescência Celular/efeitos da radiação , Radiação Ionizante , Baço/metabolismo , Baço/efeitos da radiação , Linfócitos T/imunologia , Linfócitos T/efeitos da radiação , Animais , Antivirais/uso terapêutico , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/virologia , Proliferação de Células/efeitos da radiação , Células Cultivadas , Senescência Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Ganciclovir/uso terapêutico , Vírus da Coriomeningite Linfocítica/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Fenótipo , Rejuvenescimento/fisiologia , Baço/virologiaRESUMO
Lassa virus (LASV) causes Lassa hemorrhagic fever in humans and poses a significant threat to public health in West Africa. Current therapeutic treatments for Lassa fever are limited, making the development of novel countermeasures an urgent priority. In this study, we identified losmapimod, a p38 mitogen-activated protein kinase (MAPK) inhibitor, from 102 screened compounds as an inhibitor of LASV infection. Losmapimod exerted its inhibitory effect against LASV after p38 MAPK down-regulation, and, interestingly, had no effect on other arenaviruses capable of causing viral hemorrhagic fever. Mechanistic studies showed that losmapimod inhibited LASV entry by affecting the stable signal peptide (SSP)-GP2 subunit interface of the LASV glycoprotein, thereby blocking pH-dependent viral fusion. As an aryl heteroaryl bis-carboxyamide derivative, losmapimod represents a novel chemical scaffold with anti-LASV activity, and it provides a new lead structure for the future development of LASV fusion inhibitors.
Assuntos
Antivirais/farmacologia , Ciclopropanos/farmacologia , Vírus Lassa/efeitos dos fármacos , Piridinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Infecções por Arenaviridae/tratamento farmacológico , Arenavirus/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Reposicionamento de Medicamentos , Inibidores Enzimáticos/farmacologia , Humanos , Febre Lassa/tratamento farmacológico , Febre Lassa/virologia , Células Vero , Proteínas Virais de Fusão/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Arenaviruses are a large family of emerging enveloped negative-strand RNA viruses that include several causative agents of viral hemorrhagic fevers. For cell entry, human-pathogenic arenaviruses use different cellular receptors and endocytic pathways that converge at the level of acidified late endosomes, where the viral envelope glycoprotein mediates membrane fusion. Inhibitors of arenavirus entry hold promise for therapeutic antiviral intervention and the identification of "druggable" targets is of high priority. Using a recombinant vesicular stomatitis virus pseudotype platform, we identified the clotrimazole-derivative TRAM-34, a highly selective antagonist of the calcium-activated potassium channel KCa3.1, as a specific entry inhibitor for arenaviruses. TRAM-34 specifically blocked entry of most arenaviruses, including hemorrhagic fever viruses, but not Lassa virus and other enveloped viruses. Anti-arenaviral activity was likewise observed with the parental compound clotrimazole and the derivative senicapoc, whereas structurally unrelated KCa3.1 inhibitors showed no antiviral effect. Deletion of KCa3.1 by CRISPR/Cas9 technology did not affect the antiarenaviral effect of TRAM-34, indicating that the observed antiviral effect of clotrimazoles was independent of the known pharmacological target. The drug affected neither virus-cell attachment, nor endocytosis, suggesting an effect on later entry steps. Employing a quantitative cell-cell fusion assay that bypasses endocytosis, we demonstrate that TRAM-34 specifically inhibits arenavirus-mediated membrane fusion. In sum, we uncover a novel antiarenaviral action of clotrimazoles that currently undergo in vivo evaluation in the context of other human diseases. Their favorable in vivo toxicity profiles and stability opens the possibility to repurpose clotrimazole derivatives for therapeutic intervention against human-pathogenic arenaviruses.IMPORTANCE Emerging human-pathogenic arenaviruses are causative agents of severe hemorrhagic fevers with high mortality and represent serious public health problems. The current lack of a licensed vaccine and the limited treatment options makes the development of novel antiarenaviral therapeutics an urgent need. Using a recombinant pseudotype platform, we uncovered that clotrimazole drugs, in particular TRAM-34, specifically inhibit cell entry of a range of arenaviruses, including important emerging human pathogens, with the exception of Lassa virus. The antiviral effect was independent of the known pharmacological drug target and involved inhibition of the unusual membrane fusion mechanism of arenaviruses. TRAM-34 and its derivatives currently undergo evaluation against a number of human diseases and show favorable toxicity profiles and high stability in vivo Our study provides the basis for further evaluation of clotrimazole derivatives as antiviral drug candidates. Their advanced stage of drug development will facilitate repurposing for therapeutic intervention against human-pathogenic arenaviruses.
Assuntos
Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Clotrimazol/farmacologia , Fusão de Membrana/efeitos dos fármacos , Células A549 , Animais , Infecções por Arenaviridae/tratamento farmacológico , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Endocitose/efeitos dos fármacos , Células HEK293 , Células HeLa , Febres Hemorrágicas Virais/tratamento farmacológico , Febres Hemorrágicas Virais/virologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Vírus Lassa/efeitos dos fármacos , Células Vero , Proteínas do Envelope Viral/metabolismo , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacosRESUMO
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.
Assuntos
Infecções por Arenaviridae/imunologia , Neoplasias do Colo/imunologia , Interferon Tipo I/farmacologia , Coriomeningite Linfocítica/imunologia , Melanoma Experimental/imunologia , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Animais , Antivirais/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/metabolismo , Infecções por Arenaviridae/virologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/virologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Interferon gama/metabolismo , Coriomeningite Linfocítica/tratamento farmacológico , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/fisiologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/virologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
The family Arenaviridae currently comprises over 20 viral species, each of them associated with a main rodent species as the natural reservoir and in one case possibly phyllostomid bats. Moreover, recent findings have documented a divergent group of arenaviruses in captive alethinophidian snakes. Human infections occur through mucosal exposure to aerosols or by direct contact of abraded skin with infectious materials. Arenaviruses merit interest both as highly tractable experimental model systems to study acute and persistent infections and as clinically important human pathogens including Lassa (LASV) and Junin (JUNV) viruses, the causative agents of Lassa and Argentine hemorrhagic fevers (AHFs), respectively, for which there are no FDA-licensed vaccines, and current therapy is limited to an off-label use of ribavirin (Rib) that has significant limitations. Arenaviruses are enveloped viruses with a bi-segmented negative strand (NS) RNA genome. Each genome segment, L (ca 7.3 kb) and S (ca 3.5 kb), uses an ambisense coding strategy to direct the synthesis of two polypeptides in opposite orientation, separated by a noncoding intergenic region (IGR). The S genomic RNA encodes the virus nucleoprotein (NP) and the precursor (GPC) of the virus surface glycoprotein that mediates virus receptor recognition and cell entry via endocytosis. The L genome RNA encodes the viral RNA-dependent RNA polymerase (RdRp, or L polymerase) and the small (ca 11 kDa) RING finger protein Z that has functions of a bona fide matrix protein including directing virus budding. Arenaviruses were thought to be relatively stable genetically with intra- and interspecies amino acid sequence identities of 90-95 % and 44-63 %, respectively. However, recent evidence has documented extensive arenavirus genetic variability in the field. Moreover, dramatic phenotypic differences have been documented among closely related LCMV isolates. These data provide strong evidence of viral quasispecies involvement in arenavirus adaptability and pathogenesis. Here, we will review several aspects of the molecular biology of arenaviruses, phylogeny and evolution, and quasispecies dynamics of arenavirus populations for a better understanding of arenavirus pathogenesis, as well as for the development of novel antiviral strategies to combat arenavirus infections.
Assuntos
Infecções por Arenaviridae/virologia , Arenavirus/genética , Evolução Molecular , Animais , Antivirais/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Arenavirus/classificação , Arenavirus/efeitos dos fármacos , Arenavirus/fisiologia , Variação Genética , Genoma Viral , Humanos , Filogenia , Replicação ViralRESUMO
BACKGROUND: In 2008 a nosocomial outbreak of five cases of viral hemorrhagic fever due to a novel arenavirus, Lujo virus, occurred in Johannesburg, South Africa. Lujo virus is only the second pathogenic arenavirus, after Lassa virus, to be recognized in Africa and the first in over 40 years. Because of the remote, resource-poor, and often politically unstable regions where Lassa fever and other viral hemorrhagic fevers typically occur, there have been few opportunities to undertake in-depth study of their clinical manifestations, transmission dynamics, pathogenesis, or response to treatment options typically available in industrialized countries. METHODS AND FINDINGS: We describe the clinical features of five cases of Lujo hemorrhagic fever and summarize their clinical management, as well as providing additional epidemiologic detail regarding the 2008 outbreak. Illness typically began with the abrupt onset of fever, malaise, headache, and myalgias followed successively by sore throat, chest pain, gastrointestinal symptoms, rash, minor hemorrhage, subconjunctival injection, and neck and facial swelling over the first week of illness. No major hemorrhage was noted. Neurological signs were sometimes seen in the late stages. Shock and multi-organ system failure, often with evidence of disseminated intravascular coagulopathy, ensued in the second week, with death in four of the five cases. Distinctive treatment components of the one surviving patient included rapid commencement of the antiviral drug ribavirin and administration of HMG-CoA reductase inhibitors (statins), N-acetylcysteine, and recombinant factor VIIa. CONCLUSIONS: Lujo virus causes a clinical syndrome remarkably similar to Lassa fever. Considering the high case-fatality and significant logistical impediments to controlled treatment efficacy trials for viral hemorrhagic fever, it is both logical and ethical to explore the use of the various compounds used in the treatment of the surviving case reported here in future outbreaks. Clinical observations should be systematically recorded to facilitate objective evaluation of treatment efficacy. Due to the risk of secondary transmission, viral hemorrhagic fever precautions should be implemented for all cases of Lujo virus infection, with specialized precautions to protect against aerosols when performing enhanced-risk procedures such as endotracheal intubation.
Assuntos
Antivirais/uso terapêutico , Infecções por Arenaviridae/patologia , Infecção Hospitalar/patologia , Surtos de Doenças , Febres Hemorrágicas Virais/patologia , Lujo virus/isolamento & purificação , Acetilcisteína/uso terapêutico , Adulto , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/epidemiologia , Infecções por Arenaviridae/virologia , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/virologia , Fator VIIa/uso terapêutico , Evolução Fatal , Feminino , Febres Hemorrágicas Virais/tratamento farmacológico , Febres Hemorrágicas Virais/epidemiologia , Febres Hemorrágicas Virais/virologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Febre Lassa/patologia , Vírus Lassa/isolamento & purificação , Lujo virus/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/uso terapêutico , Ribavirina/uso terapêutico , África do Sul/epidemiologiaRESUMO
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.
Assuntos
Infecções por Arenaviridae/tratamento farmacológico , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/imunologia , Interleucina-2/administração & dosagem , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Anticorpos/administração & dosagem , Anticorpos/farmacologia , Antivirais/administração & dosagem , Antivirais/farmacologia , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/virologia , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Doença Crônica , Sinergismo Farmacológico , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/farmacologia , Imunoterapia , Interleucina-2/farmacologia , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Ativação Linfocitária , Vírus da Coriomeningite Linfocítica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Carga Viral/efeitos dos fármacos , Viremia/tratamento farmacológico , Viremia/imunologia , Viremia/virologiaRESUMO
Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22- and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4(+) T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.
Assuntos
Anticorpos Antivirais/sangue , Antioxidantes/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/imunologia , Imunidade Humoral/efeitos dos fármacos , Vírus da Coriomeningite Linfocítica/imunologia , Metaloporfirinas/farmacologia , Doença Aguda , Animais , Anticorpos Antivirais/imunologia , Células Produtoras de Anticorpos/efeitos dos fármacos , Células Produtoras de Anticorpos/imunologia , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Infecções por Arenaviridae/metabolismo , Proteína 11 Semelhante a Bcl-2 , Células da Medula Óssea/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Citoproteção , Feminino , Sequestradores de Radicais Livres/farmacologia , Memória Imunológica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Arenaviruses include lethal human pathogens which pose serious public health threats. So far, no FDA approved vaccines are available against arenavirus infections, and therapeutic options are limited, making the identification of novel drug targets for the development of efficacious therapeutics an urgent need. Arenaviruses are comprised of two RNA genome segments and four proteins, the polymerase L, the envelope glycoprotein GP, the matrix protein Z, and the nucleoprotein NP. A crucial step in the arenavirus life-cycle is the biosynthesis and maturation of the GP precursor (GPC) by cellular signal peptidases and the cellular enzyme Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P) yielding a tripartite mature GP complex formed by GP1/GP2 and a stable signal peptide (SSP). GPC cleavage by SKI-1/S1P is crucial for fusion competence and incorporation of mature GP into nascent budding virion particles. In a first part of our review, we cover basic aspects and newer developments in the biosynthesis of arenavirus GP and its molecular interaction with SKI-1/S1P. A second part will then highlight the potential of SKI-1/S1P-mediated processing of arenavirus GPC as a novel target for therapeutic intervention to combat human pathogenic arenaviruses.
Assuntos
Arenavirus/metabolismo , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Proteínas do Envelope Viral/biossíntese , Sequência de Aminoácidos , Antivirais/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/metabolismo , Infecções por Arenaviridae/virologia , Arenavirus/efeitos dos fármacos , Arenavirus/patogenicidade , Glicosilação , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Humanos , Pró-Proteína Convertases/antagonistas & inibidores , Sinais Direcionadores de Proteínas , Proteólise , Pirrolidinas/farmacologia , Receptores de Superfície Celular/metabolismo , Proteínas do Envelope Viral/química , Montagem de Vírus , Ligação ViralRESUMO
Arenaviruses are a family of enveloped negative-stranded RNA viruses that can cause severe human disease ranging from encephalitis symptoms to fulminant hemorrhagic fever. The bisegmented RNA genome encodes four polypeptides: the nucleoprotein NP, the surface glycoprotein GP, the polymerase L, and the RING finger protein Z. Although it is the smallest arenavirus protein with a length of 90 to 99 amino acids and a molecular weight of approx. 11 kDa, the Z protein has multiple functions in the viral life cycle including (i) regulation of viral RNA synthesis, (ii) orchestration of viral assembly and budding, (iii) interaction with host cell proteins, and (iv) interferon antagonism. In this review, we summarize our current understanding of the structural and functional role of the Z protein in the arenavirus replication cycle.
Assuntos
Arenavirus/fisiologia , Proteínas Virais/metabolismo , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/virologia , Genoma Viral , Humanos , Domínios RING Finger , Interferência de RNA , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteínas Virais/genética , Vírion/ultraestrutura , Replicação ViralRESUMO
Arenaviruses are a large group of emerging viruses including several causative agents of severe hemorrhagic fevers with high mortality in man. Considering the number of people affected and the currently limited therapeutic options, novel efficacious therapeutics against arenaviruses are urgently needed. Over the past decade, significant advances in knowledge about the basic virology of arenaviruses have been accompanied by the development of novel therapeutics targeting different steps of the arenaviral life cycle. High-throughput, small-molecule screens identified potent and broadly active inhibitors of arenavirus entry that were instrumental for the dissection of unique features of arenavirus fusion. Novel inhibitors of arenavirus replication have been successfully tested in animal models and hold promise for application in humans. Late in the arenavirus life cycle, the proteolytic processing of the arenavirus envelope glycoprotein precursor and cellular factors critically involved virion assembly and budding provide further promising 'druggable' targets for novel therapeutics to combat human arenavirus infection.
Assuntos
Antivirais/farmacologia , Antivirais/uso terapêutico , Infecções por Arenaviridae/tratamento farmacológico , Arenaviridae/efeitos dos fármacos , Arenaviridae/fisiologia , Descoberta de Drogas , Humanos , Proteínas do Envelope Viral , Proteínas Virais de Fusão/antagonistas & inibidores , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Arenaviruses are enveloped, bipartite negative single-stranded RNA viruses that can cause a wide spectrum of disease in humans and experimental animals including hemorrhagic fever. The majority of these viruses are rodent-borne and the arenavirus family can be divided into two groups: the Lassa-Lymphocytic choriomeningitis serocomplex and the Tacaribe serocomplex. Arenavirus-induced disease may include characteristic symptoms ranging from fever, malaise, body aches, petechiae, dehydration, hemorrhage, organ failure, shock, and in severe cases death. Currently, there are few prophylactic and therapeutic treatments available for arenavirus-induced symptoms. Supportive care and ribavirin remain the predominant strategies for treating most of the arenavirus-induced diseases. Therefore, efficacy testing of novel therapeutic and prophylactic strategies in relevant animal models is necessary. Because of the potential for person-to-person spread, the ability to cause lethal or debilitating disease in humans, limited treatment options, and potential as a bio-weapon, the development of prophylactics and therapeutics is essential. This article reviews the current arenavirus animal models and prophylactic and therapeutic strategies under development to treat arenavirus infection.
Assuntos
Antivirais/administração & dosagem , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/patologia , Arenavirus/patogenicidade , Modelos Animais de Doenças , Animais , Quimioprevenção/métodos , Resultado do TratamentoRESUMO
Persistent viral infections often overburden the immune system and are a major cause of disease in humans. During many persistent infections, antiviral T cells are maintained in a state of immune exhaustion characterized by diminished effector and helper functions. In mammalian systems, an extensive immune regulatory network exists to limit unwanted, potentially fatal immunopathology by inducing T cell exhaustion. However, this regulatory network at times overprotects the host and fosters viral persistence by severely dampening adaptive immune responsiveness. Importantly, recent studies have shown that T cell exhaustion is mediated in part by host immunoregulatory pathways (e.g., programmed death 1 [PD-1], interleukin 10 [IL-10]) and that therapeutic blockade of these pathways either before or during persistent infection can promote viral clearance. Transforming growth factor beta (TGF-ß) is another immunosuppressive cytokine known to impede both self- and tumor-specific T cells, but its role in regulating antiviral immunity is not entirely understood. In this study, we inhibited TGF-ß with three potent antagonists to determine whether neutralization of this regulatory molecule is a viable approach to control a persistent viral infection. Our results revealed that these inhibitors modestly elevate the number of antiviral T cells following infection with a persistent variant of lymphocytic choriomeningitis virus (LCMV) but have no impact on viral clearance. These data suggest that therapeutic neutralization of TGF-ß is not an efficacious means to promote clearance of a persistent viral infection.
Assuntos
Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/imunologia , Linfócitos T CD8-Positivos/imunologia , Fatores Imunológicos/administração & dosagem , Vírus da Coriomeningite Linfocítica/imunologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Infecções por Arenaviridae/virologia , Linfócitos T CD4-Positivos/imunologia , Doença Crônica , Citocinas/biossíntese , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Resultado do TratamentoRESUMO
Members of the Arenaviridae family are a threat to public health and can cause meningitis and hemorrhagic fever, and yet treatment options remain limited by a lack of effective antivirals. In this study, we found that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) complementary to viral genomic RNA were effective in reducing arenavirus replication in cell cultures and in vivo. PPMO complementary to the Junín virus genome were designed to interfere with viral RNA synthesis or translation or both. However, only PPMO designed to potentially interfere with translation were effective in reducing virus replication. PPMO complementary to sequences that are highly conserved across the arenaviruses and located at the 5' termini of both genomic segments were effective against Junín virus, Tacaribe virus, Pichinde virus, and lymphocytic choriomeningitis virus (LCMV)-infected cell cultures and suppressed viral titers in the livers of LCMV-infected mice. These results suggest that arenavirus 5' genomic termini represent promising targets for pan-arenavirus antiviral therapeutic development.
Assuntos
Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Morfolinos/farmacologia , Peptídeos/farmacologia , Animais , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/virologia , Arenavirus/genética , Arenavirus/crescimento & desenvolvimento , Arenavirus do Novo Mundo/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Genoma Viral , Vírus Junin/efeitos dos fármacos , Vírus da Coriomeningite Linfocítica/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Vírus Pichinde/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , RNA Viral/genética , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Hemophagocytic lymphohistiocytosis (HLH) is an immunodysregulatory disorder for which more effective treatments are needed. The macrolide rapamycin has immunosuppressive properties, making it an attractive candidate for controlling the aberrant T cell activation that occurs in HLH. To investigate its therapeutic potential, we used rapamycin to treat Lymphocytic Choriomeningitis Virus (LCMV)-infected perforin-deficient (Prf1(-/-)) mice according to a well-established model of HLH. At the regimens tested, rapamycin did not improve weight loss, splenomegaly, hemophagocytosis, cytopenias, or proinflammatory cytokine production in LCMV-infected Prf1(-/-) animals. Thus, single agent rapamycin appears ineffective in treating the clinical and laboratory manifestations of LCMV-induced HLH.