ABSTRACT
Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus associated with a robust systemic infection and an acute inflammatory rheumatic disease. A high fiber diet has been widely promoted for its ability to ameliorate inflammatory diseases. Fiber is fermented in the gut into short chain fatty acids such as acetate, propionate, and butyrate, which enter the circulation providing systemic anti-inflammatory activities. Herein we show that mice fed a high fiber diet show a clear exacerbation of CHIKV arthropathy, with increased edema and neutrophil infiltrates. RNA-Seq analyses illustrated that a high fiber diet, in this setting, promoted a range of pro-neutrophil responses including Th17/IL-17. Gene Set Enrichment Analyses demonstrated significant similarities with mouse models of inflammatory psoriasis and significant depression of macrophage resolution phase signatures in the CHIKV arthritic lesions from mice fed a high fiber diet. Supplementation of the drinking water with butyrate also increased edema after CHIKV infection. However, the mechanisms involved were different, with modulation of AP-1 and NF-κB responses identified, potentially implicating deoptimization of endothelial barrier repair. Thus, neither fiber nor short chain fatty acids provided benefits in this acute infectious disease setting, which is characterized by widespread viral cytopathic effects and a need for tissue repair.
Subject(s)
Butyrates/adverse effects , Chikungunya Fever/immunology , Chikungunya virus/physiology , Dietary Fiber/adverse effects , Inflammation/etiology , Neutrophils/immunology , Rheumatic Diseases/etiology , Animals , Butyrates/administration & dosage , Chikungunya Fever/complications , Diet , Dietary Fiber/administration & dosage , Disease Models, Animal , Disease Progression , Edema , Humans , Joint Diseases , Mice , Mice, Inbred C57BL , Neutrophil InfiltrationABSTRACT
SerpinB2 (plasminogen activator inhibitor type 2) has been called the "undecided serpin" with no clear consensus on its physiological role, although it is well described as an inhibitor of urokinase plasminogen activator (uPA). In macrophages, pro-inflammatory stimuli usually induce SerpinB2; however, expression is constitutive in Gata6+ large peritoneal macrophages (LPM). Interrogation of expression data from human macrophages treated with a range of stimuli using a new bioinformatics tool, CEMiTool, suggested that SerpinB2 is most tightly co- and counter-regulated with genes associated with cell movement. Using LPM from SerpinB2-/- and SerpinB2R380A (active site mutant) mice, we show that migration on Matrigel was faster than for their wild-type controls. Confocal microscopy illustrated that SerpinB2 and F-actin staining overlapped in focal adhesions and lamellipodia. Genes associated with migration and extracellular matrix interactions were also identified by RNA-Seq analysis of migrating RPM from wild-type and SerpinB2R380A mice. Subsequent gene set enrichment analyses (GSEA) suggested SerpinB2 counter-regulates many Gata6-regulated genes associated with migration. These data argue that the role of SerpinB2 in macrophages is inhibition of uPA-mediated plasmin generation during cell migration. GSEA also suggested that SerpinB2 expression (likely via ensuing modulation of uPA-receptor/integrin signaling) promotes the adoption of a resolution phase signature.
Subject(s)
Cell Movement , Macrophages, Peritoneal/metabolism , Plasminogen Activator Inhibitor 2/metabolism , Animals , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Macrophages, Peritoneal/cytology , Mice , Mice, Knockout , Plasminogen Activator Inhibitor 2/genetics , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
SerpinB2, also known as plasminogen activation inhibitor type 2 (PAI-2), is classically viewed as an inhibitor of fibrinolysis. However, we show herein a distinct, hitherto unrecognized role for SerpinB2 in hemostasis. Mice deficient in SerpinB2 expression and mice with an active site mutation in SerpinB2, both showed significant reductions in tail bleeding times. This hemostatic phenotype was associated with platelets, with SerpinB2 and SerpinB2-urokinase complexes clearly present in platelet fractions, and immunohistochemistry of blood clots suggesting SerpinB2 is associated with platelet aggregates. Thromboelastography illustrated faster onset of clot formation in blood from SerpinB2 deficient mice, whereas clotting of platelet-free plasma was unaffected. The results appear consistent with the low circulating SerpinB2 levels and hypercoagulation seen during pre-eclampsia; however, SerpinB2 was not detected in human platelets.
Subject(s)
Bleeding Time , Blood Coagulation/genetics , Blood Platelets/metabolism , Plasminogen Activator Inhibitor 2/deficiency , Platelet Activation , Animals , Biomarkers , Female , Immunohistochemistry , Mice , Mice, Knockout , Pregnancy , Thrombosis , Urokinase-Type Plasminogen ActivatorABSTRACT
Chikungunya virus (CHIKV) belongs to a group of mosquito-borne alphaviruses associated with acute and chronic arthropathy, with peripheral and limb joints most commonly affected. Using a mouse model of CHIKV infection and arthritic disease, we show that CHIKV replication and the ensuing foot arthropathy were dramatically reduced when mice were housed at 30°C, rather than the conventional 22°C. The effect was not associated with a detectable fever, but was dependent on type I interferon responses. Bioinformatics analyses of RNA-Seq data after injection of poly(I:C)/jetPEI suggested the unfolded protein response and certain type I interferon responses are promoted when feet are slightly warmer. The ambient temperature thus appears able profoundly to effect anti-viral activity in the periphery, with clear consequences for alphaviral replication and the ensuing arthropathy. These observations may provide an explanation for why alphaviral arthropathies are largely restricted to joints of the limbs and the extremities.
Subject(s)
Alphavirus Infections/immunology , Alphavirus Infections/virology , Arthritis, Experimental/immunology , Arthritis, Experimental/virology , Arthritis, Infectious/immunology , Arthritis, Infectious/virology , Interferon Type I/metabolism , Alphavirus Infections/pathology , Animals , Arthritis, Experimental/pathology , Arthritis, Infectious/pathology , Chikungunya Fever/immunology , Chikungunya Fever/pathology , Chikungunya Fever/virology , Chikungunya virus/immunology , Chikungunya virus/pathogenicity , Chikungunya virus/physiology , Female , Foot , Host-Pathogen Interactions/immunology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Ross River virus/immunology , Ross River virus/pathogenicity , Ross River virus/physiology , Temperature , Viral Load , Virus Replication/immunology , Virus Replication/physiologyABSTRACT
Chikungunya virus (CHIKV) is an arthritogenic alphavirus causing epidemics of acute and chronic arthritic disease. Herein we describe a comprehensive RNA-Seq analysis of feet and lymph nodes at peak viraemia (day 2 post infection), acute arthritis (day 7) and chronic disease (day 30) in the CHIKV adult wild-type mouse model. Genes previously shown to be up-regulated in CHIKV patients were also up-regulated in the mouse model. CHIKV sequence information was also obtained with up to ≈8% of the reads mapping to the viral genome; however, no adaptive viral genome changes were apparent. Although day 2, 7 and 30 represent distinct stages of infection and disease, there was a pronounced overlap in up-regulated host genes and pathways. Type I interferon response genes (IRGs) represented up to ≈50% of up-regulated genes, even after loss of type I interferon induction on days 7 and 30. Bioinformatic analyses suggested a number of interferon response factors were primarily responsible for maintaining type I IRG induction. A group of genes prominent in the RNA-Seq analysis and hitherto unexplored in viral arthropathies were granzymes A, B and K. Granzyme A-/- and to a lesser extent granzyme K-/-, but not granzyme B-/-, mice showed a pronounced reduction in foot swelling and arthritis, with analysis of granzyme A-/- mice showing no reductions in viral loads but reduced NK and T cell infiltrates post CHIKV infection. Treatment with Serpinb6b, a granzyme A inhibitor, also reduced arthritic inflammation in wild-type mice. In non-human primates circulating granzyme A levels were elevated after CHIKV infection, with the increase correlating with viral load. Elevated granzyme A levels were also seen in a small cohort of human CHIKV patients. Taken together these results suggest granzyme A is an important driver of arthritic inflammation and a potential target for therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00281294.
Subject(s)
Arthritis/virology , Chikungunya Fever/genetics , Chikungunya Fever/immunology , Granzymes/immunology , Inflammation/virology , Animals , Chikungunya virus , Disease Models, Animal , Granzymes/analysis , Granzymes/biosynthesis , Humans , Immunohistochemistry , Macaca fascicularis , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/analysis , TranscriptomeABSTRACT
Chikungunya virus (CHIKV) is a reemerging, ordinarily mosquito-transmitted, alphavirus that occasionally produces hemorrhagic manifestations, such as nose bleed and bleeding gums, in human patients. Interferon response factor 3 and 7 deficient (IRF3/7-/-) mice, which are deficient for interferon α/ß responses, reliably develop hemorrhagic manifestations after CHIKV infection. Here we show that infectious virus was present in the oral cavity of CHIKV infected IRF3/7-/- mice, likely due to hemorrhagic lesions in the olfactory epithelium that allow egress of infected blood into the nasal, and subsequently, oral cavities. In addition, IRF3/7-/- mice were more susceptible to infection with CHIKV via intranasal and oral routes, with IRF3/7-/- mice also able to transmit virus mouse-to-mouse without an arthropod vector. Cynomolgus macaques often show bleeding gums after CHIKV infection, and analysis of saliva from several infected monkeys also revealed the presence of viral RNA and infectious virus. Furthermore, saliva samples collected from several acute CHIKV patients with hemorrhagic manifestations were found to contain viral RNA and infectious virus. Oral fluids can therefore be infectious during acute CHIKV infections, likely due to hemorrhagic manifestations in the oral/nasal cavities.
Subject(s)
Chikungunya Fever/pathology , Chikungunya virus/genetics , Saliva/virology , Adolescent , Animals , Chikungunya Fever/transmission , Chikungunya Fever/virology , Chikungunya virus/isolation & purification , Child , Disease Models, Animal , Female , Haplorhini , Humans , Interferon Regulatory Factor-3/deficiency , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-7/deficiency , Interferon Regulatory Factor-7/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Olfactory Mucosa/pathology , Olfactory Mucosa/virology , RNA, Viral/metabolism , Viral LoadABSTRACT
Foroderm is a new cutaneous delivery technology that uses high-aspect ratio, cylindrical silica microparticles, that are massaged into the skin using a 3D-printed microtextured applicator, in order to deliver payloads across the epidermis. Herein we show that this technology is effective for delivery of a non-adjuvanted, inactivated, whole-virus chikungunya virus vaccine in mice, with minimal post-vaccination skin reactions. A single topical Foroderm-based vaccination induced T cell, Th1 cytokine and antibody responses, which provided complete protection against viraemia and disease after challenge with chikungunya virus. Foroderm vaccination was shown to deliver fluorescent, virus-sized beads across the epidermis, with beads subsequently detected in draining lymph nodes. Foroderm vaccination also stimulated the egress of MHC II(+) antigen presenting cells from the skin. Foroderm thus has potential as a simple, cheap, effective, generic, needle-free technology for topical delivery of vaccines.
Subject(s)
Chikungunya Fever/prevention & control , Chikungunya virus/immunology , Drug Delivery Systems , Viral Vaccines/immunology , Administration, Cutaneous , Animals , Antibodies, Viral/blood , Disease Models, Animal , Female , Mice, Inbred Strains , Mice, Nude , T-Lymphocytes/immunology , Treatment Outcome , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/immunology , Viral Vaccines/administration & dosage , Viremia/prevention & controlABSTRACT
Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for epidemics of debilitating arthritic disease. The recent outbreak (2004-2014) resulted in an estimated 1.4-6.5 million cases, with imported cases reported in nearly 40 countries. The development of CHIKV-specific diagnostics and research tools is thus highly desirable. Herein we describe the generation and characterization of the first mAbs specific for the capsid protein (CP) of CHIKV. The antibodies recognized isolates representing the major genotypes of CHIKV, as well as several other alphaviruses, and were reactive in a range of assays including ELISA, Western blot, immunofluorescence and immunohistochemistry (IHC). We have also used the anti-CP mAb 5.5G9 in IHC studies to show that capsid antigen is persistently expressed 30 days post-infection in cells with macrophage morphology in a mouse model of chronic CHIKV disease. These antibodies may thus represent useful tools for further research, including investigations into the structure and function of CHIKV CP, and as valuable reagents for CHIKV detection in a range of settings.
Subject(s)
Antibodies, Monoclonal/immunology , Capsid Proteins/immunology , Chikungunya virus/immunology , Animals , Antibody Specificity , Blotting, Western , COS Cells , Capsid Proteins/genetics , Capsid Proteins/metabolism , Chikungunya Fever/immunology , Chikungunya Fever/virology , Chikungunya virus/genetics , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Gene Expression Regulation, Viral , Humans , Immunohistochemistry , Macrophages/metabolism , Mice , Mice, Inbred BALB CABSTRACT
The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1α become activated. PERK phosphorylates eIF2α leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1α splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2α and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.
Subject(s)
Chikungunya Fever/virology , Chikungunya virus/metabolism , Gene Expression Regulation/physiology , Unfolded Protein Response/physiology , Viral Nonstructural Proteins/metabolism , Animals , Arthritis, Infectious , Chikungunya Fever/pathology , Chikungunya virus/genetics , Chlorocebus aethiops , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum Chaperone BiP , Gene Expression Regulation/immunology , Mice , RNA Splicing , Regulatory Factor X Transcription Factors , Transcription Factors/genetics , Transcription Factors/metabolism , Tunicamycin/pharmacology , Vero Cells , Viral Nonstructural Proteins/genetics , Virus Replication , X-Box Binding Protein 1ABSTRACT
The recent epidemic of the arthritogenic alphavirus, chikungunya virus (CHIKV) has prompted a quest to understand the correlates of protection against virus and disease in order to inform development of new interventions. Herein we highlight the propensity of CHIKV infections to persist long term, both as persistent, steady-state, viraemias in multiple B cell deficient mouse strains, and as persistent RNA (including negative-strand RNA) in wild-type mice. The knockout mouse studies provided evidence for a role for T cells (but not NK cells) in viraemia suppression, and confirmed the role of T cells in arthritis promotion, with vaccine-induced T cells also shown to be arthritogenic in the absence of antibody responses. However, MHC class II-restricted T cells were not required for production of anti-viral IgG2c responses post CHIKV infection. The anti-viral cytokines, TNF and IFNγ, were persistently elevated in persistently infected B and T cell deficient mice, with adoptive transfer of anti-CHIKV antibodies unable to clear permanently the viraemia from these, or B cell deficient, mice. The NOD background increased viraemia and promoted arthritis, with B, T and NK deficient NOD mice showing high-levels of persistent viraemia and ultimately succumbing to encephalitic disease. In wild-type mice persistent CHIKV RNA and negative strand RNA (detected for up to 100 days post infection) was associated with persistence of cellular infiltrates, CHIKV antigen and stimulation of IFNα/ß and T cell responses. These studies highlight that, secondary to antibodies, several factors are involved in virus control, and suggest that chronic arthritic disease is a consequence of persistent, replicating and transcriptionally active CHIKV RNA.
Subject(s)
Chikungunya Fever/immunology , Chikungunya virus/immunology , Acute Disease , Animals , Antibodies, Viral/immunology , B-Lymphocytes/immunology , Chikungunya Fever/genetics , Chikungunya Fever/virology , Chronic Disease , Disease Models, Animal , Female , Killer Cells, Natural/immunology , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , RNA, Viral/analysis , T-Lymphocytes/immunologyABSTRACT
UNLABELLED: Chikungunya virus (CHIKV) is a member of a globally distributed group of arthritogenic alphaviruses that cause weeks to months of debilitating polyarthritis/arthralgia, which is often poorly managed with current treatments. Arthritic disease is usually characterized by high levels of the chemokine CCL2 and a prodigious monocyte/macrophage infiltrate. Several inhibitors of CCL2 and its receptor CCR2 are in development and may find application for treatment of certain inflammatory conditions, including autoimmune and viral arthritides. Here we used CCR2(-/-) mice to determine the effect of CCR2 deficiency on CHIKV infection and arthritis. Although there were no significant changes in viral load or RNA persistence and only marginal changes in antiviral immunity, arthritic disease was substantially increased and prolonged in CCR2(-/-) mice compared to wild-type mice. The monocyte/macrophage infiltrate was replaced in CCR2(-/-) mice by a severe neutrophil (followed by an eosinophil) infiltrate and was associated with changes in the expression levels of multiple inflammatory mediators (including CXCL1, CXCL2, granulocyte colony-stimulating factor [G-CSF], interleukin-1ß [IL-1ß], and IL-10). The loss of anti-inflammatory macrophages and their activities (e.g., efferocytosis) was also implicated in exacerbated inflammation. Clear evidence of cartilage damage was also seen in CHIKV-infected CCR2(-/-) mice, a feature not normally associated with alphaviral arthritides. Although recruitment of CCR2(+) monocytes/macrophages can contribute to inflammation, it also appears to be critical for preventing excessive pathology and resolving inflammation following alphavirus infection. Caution might thus be warranted when considering therapeutic targeting of CCR2/CCL2 for the treatment of alphaviral arthritides. IMPORTANCE: Here we describe the first analysis of viral arthritis in mice deficient for the chemokine receptor CCR2. CCR2 is thought to be central to the monocyte/macrophage-dominated inflammatory arthritic infiltrates seen after infection with arthritogenic alphaviruses such as chikungunya virus. Surprisingly, the viral arthritis caused by chikungunya virus in CCR2-deficient mice was more severe, prolonged, and erosive and was neutrophil dominated, with viral replication and persistence not being significantly affected. Monocytes/macrophages recruited by CCL2 thus also appear to be important for both preventing even worse pathology mediated by neutrophils and promoting resolution of inflammation. Caution might thus be warranted when considering the use of therapeutic agents that target CCR2/CCL2 or inflammatory monocytes/macrophages for the treatment of alphaviral (and perhaps other viral) arthritides. Individuals with diminished CCR2 responses (due to drug treatment or other reasons) may also be at risk of exacerbated arthritic disease following alphaviral infection.
Subject(s)
Alphavirus Infections/immunology , Arthritis/immunology , Chikungunya virus/physiology , Neutrophils/immunology , Receptors, CCR2/deficiency , Alphavirus Infections/genetics , Alphavirus Infections/virology , Animals , Arthritis/genetics , Arthritis/virology , Chikungunya Fever , Chikungunya virus/genetics , Granulocyte Colony-Stimulating Factor/immunology , Humans , Interleukin-10/immunology , Interleukin-1beta/immunology , Mice , Mice, Knockout , Neutrophil Infiltration , Receptors, CCR2/genetics , Receptors, CCR2/immunologyABSTRACT
Expression of SerpinB2 (plasminogen activator inhibitor type 2/PAI-2) by certain cancers is associated with a favorable prognosis. Although tumor-associated host tissues can express SerpinB2, no significant differences in the growth of a panel of different tumors in SerpinB2(-/-) and SerpinB2(+/+) mice were observed. SerpinB2 expression by cancer cells (via lentiviral transduction) also had no significant effect on the growth of panel of mouse and human tumor lines in vivo or in vitro. SerpinB2 expression by cancer cells did, however, significantly reduce the number of metastases in a B16 metastasis model. SerpinB2-expressing B16 cells also showed reduced migration and increased length of invadopodia-like structures, supporting the classical view that that tumor-derived SerpinB2 is inhibiting extracellular urokinase. Importantly, although SerpinB2 is usually poorly secreted, we found that SerpinB2 effectively reaches the extracellular milieu on the surface of 0.5-1 µm microparticles (MPs), where it was able to inhibit urokinase. We also provide evidence that annexins mediate the binding of SerpinB2 to phosphatidylserine, a lipid characteristically exposed on the surface of MPs. The presence of SerpinB2 on the surface of MPs provides a physiological mechanism whereby cancer cell SerpinB2 can reach the extracellular milieu and access urokinase plasminogen activator (uPA). This may then lead to inhibition of metastasis and a favorable prognosis.
Subject(s)
Melanoma, Experimental/genetics , Neoplasm Metastasis/genetics , Plasminogen Activator Inhibitor 2/biosynthesis , Animals , Cell-Derived Microparticles , Gene Expression Regulation, Neoplastic , Humans , Melanoma, Experimental/pathology , Mice , Neoplasm Metastasis/pathology , Phosphatidylserines/metabolism , Plasminogen Activator Inhibitor 2/genetics , Prognosis , Protein Binding , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
Chikungunya virus (CHIKV) recently caused the largest epidemic ever recorded for this virus involving an estimated 1.4-6.5million cases, with imported cased reported in over 40 countries. The number of monoclonal antibodies specific for this re-emerging alphavirus is currently limited. Herein we describe the generation and characterisation of five monoclonal antibodies specific for the E2 glycoprotein of CHIKV. The antibodies detected a range of CHIKV isolates in several assays including ELISA, Western blot, immunofluorescence assay (IFA) and immunohistochemistry (IHC) without evidence of cross-reactivity with other alphaviruses. Four antibodies also neutralised CHIKV in vitro, two of which provided complete protection against arthritis in a CHIKV mouse model when administered prior to infection. Given the current shortage of widely available reagents for CHIKV, these specific antibodies will be useful not only in research, but may also provide the basis for new diagnostics and treatments.
Subject(s)
Alphavirus Infections/prevention & control , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Arthritis, Experimental/prevention & control , Viral Envelope Proteins/immunology , Alphavirus Infections/immunology , Alphavirus Infections/virology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/biosynthesis , Antibodies, Viral/isolation & purification , Arthritis, Experimental/immunology , Arthritis, Experimental/virology , COS Cells , Chikungunya virus/drug effects , Chikungunya virus/immunology , Chlorocebus aethiops , Female , Humans , Hybridomas/immunology , Mice , Mice, Inbred BALB C , Vero Cells , Viral Envelope Proteins/geneticsABSTRACT
The mosquito-borne alphavirus, chikungunya virus (CHIKV), has recently reemerged, producing the largest epidemic ever recorded for this virus, with up to 6.5 million cases of acute and chronic rheumatic disease. There are currently no licensed vaccines for CHIKV and current anti-inflammatory drug treatment is often inadequate. Here we describe the isolation and characterization of two human monoclonal antibodies, C9 and E8, from CHIKV infected and recovered individuals. C9 was determined to be a potent virus neutralizing antibody and a biosensor antibody binding study demonstrated it recognized residues on intact CHIKV VLPs. Shotgun mutagenesis alanine scanning of 98 percent of the residues in the E1 and E2 glycoproteins of CHIKV envelope showed that the epitope bound by C9 included amino-acid 162 in the acid-sensitive region (ASR) of the CHIKV E2 glycoprotein. The ASR is critical for the rearrangement of CHIKV E2 during fusion and viral entry into host cells, and we predict that C9 prevents these events from occurring. When used prophylactically in a CHIKV mouse model, C9 completely protected against CHIKV viremia and arthritis. We also observed that when administered therapeutically at 8 or 18 hours post-CHIKV challenge, C9 gave 100% protection in a pathogenic mouse model. Given that targeting this novel neutralizing epitope in E2 can potently protect both in vitro and in vivo, it is likely to be an important region both for future antibody and vaccine-based interventions against CHIKV.
Subject(s)
Alphavirus Infections/prevention & control , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Chikungunya virus/immunology , Viral Envelope Proteins/immunology , Alphavirus Infections/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/isolation & purification , Antibodies, Viral/administration & dosage , Antibodies, Viral/isolation & purification , Chikungunya Fever , Disease Models, Animal , Epitope Mapping , Humans , Immunization, Passive , Mice , Mice, Inbred C57BL , Treatment OutcomeABSTRACT
SerpinB2, also known as plasminogen activator inhibitor type 2, is a major product of activated monocytes/macrophages and is often strongly induced during infection and inflammation; however, its physiological function remains somewhat elusive. Herein we show that SerpinB2 is induced in peripheral blood mononuclear cells following infection of pigtail macaques with CCR5-utilizing (macrophage-tropic) SIVmac239, but not the rapidly pathogenic CXCR4-utilizing (T cell-tropic) SHIVmn229. To investigate the role of SerpinB2 in lentiviral infections, SerpinB2(-/-) mice were infected with EcoHIV, a chimeric HIV in which HIV gp120 has been replaced with gp80 from ecotropic murine leukemia virus. EcoHIV infected SerpinB2(-/-) mice produced significantly lower anti-gag IgG1 antibody titres than infected SerpinB2(+/+) mice, and showed slightly delayed clearance of EcoHIV. Analyses of published microarray studies showed significantly higher levels of SerpinB2 mRNA in monocytes from HIV-1 infected patients when compared with uninfected controls, as well as a significant negative correlation between SerpinB2 and T-bet mRNA levels in peripheral blood mononuclear cells. These data illustrate that SerpinB2 can be induced by lentiviral infection in vivo and support the emerging notion that a physiological role of SerpinB2 is modulation of Th1/Th2 responses.
Subject(s)
Lentivirus Infections/immunology , Plasminogen Activator Inhibitor 2/metabolism , Th1 Cells/immunology , Th2 Cells/immunology , Animals , Cell Movement , Gene Expression Regulation , HIV Infections/genetics , HIV Infections/immunology , HIV Infections/virology , HIV-1/physiology , Humans , Immunoglobulin G/immunology , Lentivirus Infections/genetics , Lentivirus Infections/pathology , Lentivirus Infections/virology , Macaca nemestrina/immunology , Macaca nemestrina/virology , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Plasminogen Activator Inhibitor 2/deficiency , Plasminogen Activator Inhibitor 2/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Simian Immunodeficiency Virus/physiology , Virus Replication/physiologyABSTRACT
The emerging arthritogenic, mosquito-borne chikungunya virus (CHIKV) causes severe disease in humans and represents a serious public health threat in countries where Aedes spp mosquitoes are present. This study describes for the first time the successful production of CHIKV virus-like particles (VLPs) in insect cells using recombinant baculoviruses. This well-established expression system is rapidly scalable to volumes required for epidemic responses and proved well suited for processing of CHIKV glycoproteins and production of enveloped VLPs. Herein we show that a single immunization with 1 µg of non-adjuvanted CHIKV VLPs induced high titer neutralizing antibody responses and provided complete protection against viraemia and joint inflammation upon challenge with the Réunion Island CHIKV strain in an adult wild-type mouse model of CHIKV disease. CHIKV VLPs produced in insect cells using recombinant baculoviruses thus represents as a new, safe, non-replicating and effective vaccine candidate against CHIKV infections.
Subject(s)
Chikungunya virus/immunology , Vaccines, Virus-Like Particle/immunology , Viral Vaccines/immunology , Alphavirus Infections/immunology , Alphavirus Infections/prevention & control , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Baculoviridae/genetics , Cell Line , Chikungunya Fever , Chikungunya virus/genetics , Disease Models, Animal , Female , Genetic Vectors , Mice , Mice, Inbred C57BL , Spodoptera , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , Vaccines, Synthetic/isolation & purification , Vaccines, Virus-Like Particle/administration & dosage , Vaccines, Virus-Like Particle/isolation & purification , Viral Vaccines/administration & dosage , Viral Vaccines/isolation & purificationABSTRACT
Chikungunya virus (CHIKV) infections can produce severe disease and mortality. Here we show that CHIKV infection of adult mice deficient in interferon response factors 3 and 7 (IRF3/7(-/-)) is lethal. Mortality was associated with undetectable levels of alpha/beta interferon (IFN-α/ß) in serum, â¼50- and â¼10-fold increases in levels of IFN-γ and tumor necrosis factor (TNF), respectively, increased virus replication, edema, vasculitis, hemorrhage, fever followed by hypothermia, oliguria, thrombocytopenia, and raised hematocrits. These features are consistent with hemorrhagic shock and were also evident in infected IFN-α/ß receptor-deficient mice. In situ hybridization suggested CHIKV infection of endothelium, fibroblasts, skeletal muscle, mononuclear cells, chondrocytes, and keratinocytes in IRF3/7(-/-) mice; all but the latter two stained positive in wild-type mice. Vaccination protected IRF3/7(-/-) mice, suggesting that defective antibody responses were not responsible for mortality. IPS-1- and TRIF-dependent pathways were primarily responsible for IFN-α/ß induction, with IRF7 being upregulated >100-fold in infected wild-type mice. These studies suggest that inadequate IFN-α/ß responses following virus infection can be sufficient to induce hemorrhagic fever and shock, a finding with implications for understanding severe CHIKV disease and dengue hemorrhagic fever/dengue shock syndrome.
Subject(s)
Alphavirus Infections/immunology , Alphavirus Infections/prevention & control , Chikungunya virus/pathogenicity , Interferon Regulatory Factor-3/physiology , Interferon Regulatory Factor-7/physiology , Adaptor Proteins, Vesicular Transport/deficiency , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/physiology , Alphavirus Infections/pathology , Animals , Chikungunya Fever , Chikungunya virus/immunology , Chikungunya virus/physiology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Interferon Regulatory Factor-3/deficiency , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-7/deficiency , Interferon Regulatory Factor-7/genetics , Interferon-alpha/biosynthesis , Interferon-alpha/pharmacology , Interferon-beta/biosynthesis , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/deficiency , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/physiology , Receptor, Interferon alpha-beta/deficiency , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/physiology , Shock, Hemorrhagic/immunology , Shock, Hemorrhagic/prevention & control , Virus Replication/drug effectsABSTRACT
OBJECTIVE: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes a chronic debilitating polyarthralgia/polyarthritis, for which current treatments are often inadequate. To assess whether new drugs being developed for rheumatoid arthritis (RA) might find utility in the treatment of alphaviral arthritides, we sought to determine whether the inflammatory gene expression signature of CHIKV arthritis shows any similarities with RA or collagen-induced arthritis (CIA), a mouse model of RA. METHODS: Using a recently developed animal model of CHIKV arthritis in adult wild-type mice, we generated a consensus CHIKV arthritis gene expression signature, which was used to interrogate publicly available microarray studies of RA and CIA. Pathway analyses were then performed using the overlapping gene signatures. RESULTS: Gene set enrichment analysis showed that there was a highly significant overlap in the differentially expressed genes in the CHIKV arthritis model and in RA. This concordance also increased with the severity of RA, as measured by the inflammation score. A highly significant overlap was also seen between CHIKV arthritis and CIA. Pathway analysis revealed that the overlap between these arthritides was spread over a range of different inflammatory processes. Involvement of T cells and interferon-γ (IFNγ) in CHIKV arthritis was confirmed in studies of MHCII-deficient mice and IFNγ-deficient mice, respectively. CONCLUSION: These results suggest that RA, a chronic autoimmune arthritis, and CHIKV disease, usually a self-limiting viral arthropathy, share multiple inflammatory processes. New drugs and biologic therapies being developed for RA may thus find application in the treatment of alphaviral arthritides.
Subject(s)
Alphavirus Infections , Arthritis, Infectious , Arthritis, Rheumatoid , Chikungunya virus/immunology , Transcriptome/immunology , Alphavirus Infections/complications , Alphavirus Infections/genetics , Alphavirus Infections/immunology , Animals , Arthritis, Infectious/genetics , Arthritis, Infectious/immunology , Arthritis, Infectious/virology , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/virology , Asia , Chikungunya Fever , Disease Models, Animal , Gene Regulatory Networks/immunology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Reunion , Synovial Membrane/immunologyABSTRACT
Skin cancer is the most prevalent cancer worldwide and is primarily caused by chronic UV exposure. Here, we describe the topical field-directed treatment of SKH1/hr mice with UVB-damaged skin with ingenol mebutate, a new topical drug shown to be effective for the treatment of actinic keratosis (AK). Application of 0.05% ingenol mebutate gel to photo-damaged skin resulted in a ≈70% reduction in the number of skin lesions that subsequently emerged compared with placebo treatment. Ingenol mebutate treatment also reduced the number of mutant p53 keratinocyte patches by ≈70%. The treatment resulted in epidermal cell death, acute inflammation, recruitment of neutrophils, hemorrhage, and eschar formation, all of which resolved over several weeks. Ingenol mebutate field-directed treatment might thus find utility in the removal of subclinical precancerous cells from UV-damaged skin. Field-directed treatment may be particularly suitable for patients who have AKs surrounded by UV-damaged skin.
Subject(s)
Diterpenes/therapeutic use , Keratinocytes/metabolism , Keratinocytes/pathology , Keratosis, Actinic/drug therapy , Precancerous Conditions/drug therapy , Skin/radiation effects , Tumor Suppressor Protein p53/metabolism , Ultraviolet Rays/adverse effects , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Carcinoma, Squamous Cell/prevention & control , Disease Models, Animal , Diterpenes/pharmacology , Dose-Response Relationship, Drug , Keratinocytes/drug effects , Keratosis, Actinic/pathology , Male , Mice , Mice, Hairless , Mutation/genetics , Neoplasms, Radiation-Induced/prevention & control , Precancerous Conditions/pathology , Skin/drug effects , Skin/pathology , Skin Neoplasms/prevention & control , Tumor Suppressor Protein p53/geneticsABSTRACT
Chikungunya virus, a mosquito-borne alphavirus, recently caused the largest epidemic ever seen for this virus. Chikungunya disease primarily manifests as a painful and debilitating arthralgia/arthritis, and no effective drug or vaccine is currently available. Here we describe a recombinant chikungunya virus vaccine comprising a non-replicating complex adenovirus vector encoding the structural polyprotein cassette of chikungunya virus. A single immunisation with this vaccine consistently induced high titres of anti-chikungunya virus antibodies that neutralised both an old Asian isolate and a Réunion Island isolate from the recent epidemic. The vaccine also completely protected mice against viraemia and arthritic disease caused by both virus isolates.