The Ebola virus soluble glycoprotein contributes to viral pathogenesis by activating the MAP kinase signaling pathway

Ebola virus (EBOV) expresses three different glycoproteins (GPs) from its GP gene. The primary product, soluble GP (sGP), is secreted in abundance during infection. EBOV sGP has been discussed as a potential pathogenicity factor, however, little is known regarding its functional role. Here, we analyzed the role of sGP in vitro and in vivo. We show that EBOV sGP has two different functions that contribute to infectivity in tissue culture. EBOV sGP increases the uptake of virus particles into late endosomes in HEK293 cells, and it activates the mitogen-activated protein kinase (MAPK) signaling pathway leading to increased viral replication in Huh7 cells. Furthermore, we analyzed the role of EBOV sGP on pathogenicity using a well-established mouse model. We found an sGP-dependent significant titer increase of EBOV in the liver of infected animals. These results provide new mechanistic insights into EBOV pathogenicity and highlight EBOV sGP as a possible therapeutic target.     

Antigenic Subversion: A Novel Mechanism of Host Immune Evasion by Ebola Virus

In addition to its surface glycoprotein (GP_1,2), Ebola virus (EBOV) directs the production of large quantities of a truncated glycoprotein isoform (sGP) that is secreted into the extracellular space. The generation of secreted antigens has been studied in several viruses and suggested as a mechanism of host immune evasion through absorption of antibodies and interference with antibody-mediated clearance. However such a role has not been conclusively determined for the Ebola virus sGP. In this study, we immunized mice with DNA constructs expressing GP_1,2 and/or sGP, and demonstrate that sGP can efficiently compete for anti-GP₁₂ antibodies, but only from mice that have been immunized by sGP. We term this phenomenon “antigenic subversion”, and propose a model whereby sGP redirects the host antibody response to focus on epitopes which it shares with membrane-bound GP_1,2, thereby allowing it to absorb anti-GP_1,2 antibodies. Unexpectedly, we found that sGP can also subvert a previously immunized host''s anti-GP_1,2 response resulting in strong cross-reactivity with sGP. This finding is particularly relevant to EBOV vaccinology since it underscores the importance of eliciting robust immunity that is sufficient to rapidly clear an infection before antigenic subversion can occur. Antigenic subversion represents a novel virus escape strategy that likely helps EBOV evade host immunity, and may represent an important obstacle to EBOV vaccine design.     

Role of Ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages

Ebola virus, a member of the family Filoviridae, causes one of the most severe forms of viral hemorrhagic fever. In the terminal stages of disease, symptoms progress to hypotension, coagulation disorders, and hemorrhages, and there is prominent involvement of the mononuclear phagocytic and reticuloendothelial systems. Cells of the mononuclear phagocytic system are primary target cells and producers of inflammatory mediators. Ebola virus efficiently produces four soluble glycoproteins during infection: sGP, delta peptide (Delta-peptide), GP(1), and GP(1,2Delta). While the presence of these glycoproteins has been confirmed in blood (sGP) and in vitro systems, it is hypothesized that they are of biological relevance in pathogenesis, particularly target cell activation. To gain insight into their function, we expressed the four soluble glycoproteins in mammalian cells and purified and characterized them. The role of the transmembrane glycoprotein in the context of virus-like particles was also investigated. Primary human macrophages were treated with glycoproteins and virus-like particles and subsequently tested for activation by detection of several critical proinflammatory cytokines (tumor necrosis factor alpha, interleukin-6 [IL-6], and IL-1 beta) and the chemokine IL-8. The presentation of the glycoprotein was determined to be critical since virus-like particles, but not soluble glycoproteins, induced high levels of activation. We propose that the presentation of GP(1,2) in the rigid form such as that observed on the surface of particles is critical for initiating a sufficient signal for the activation of primary target cells. The secreted glycoproteins do not appear to play any role in exogenous activation of these cells during Ebola virus infection.     

Effects of Ebola virus glycoproteins on endothelial cell activation and barrier function

Ebola virus causes severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. Vascular instability and dysregulation are disease-decisive symptoms during severe infection. While the transmembrane glycoprotein GP(1,2) has been shown to cause endothelial cell destruction, the role of the soluble glycoproteins in pathogenesis is largely unknown; however, they are hypothesized to be of biological relevance in terms of target cell activation and/or increase of endothelial permeability. Here we show that virus-like particles (VLPs) consisting of the Ebola virus matrix protein VP40 and GP(1,2) were able to activate endothelial cells and induce a decrease in barrier function as determined by impedance spectroscopy and hydraulic conductivity measurements. In contrast, the soluble glycoproteins sGP and delta-peptide did not activate endothelial cells or change the endothelial barrier function. The VLP-induced decrease in barrier function was further enhanced by the cytokine tumor necrosis factor alpha (TNF-alpha), which is known to induce a long-lasting decrease in endothelial cell barrier function and is hypothesized to play a key role in Ebola virus pathogenesis. Surprisingly, sGP, but not delta-peptide, induced a recovery of endothelial barrier function following treatment with TNF-alpha. Our results demonstrate that Ebola virus GP(1,2) in its particle-associated form mediates endothelial cell activation and a decrease in endothelial cell barrier function. Furthermore, sGP, the major soluble glycoprotein of Ebola virus, seems to possess an anti-inflammatory role by protecting the endothelial cell barrier function.     

Ectodomain shedding of the glycoprotein GP of Ebola virus

In this study, release of abundant amounts of the Ebola virus (EBOV) surface glycoprotein GP in a soluble form from virus-infected cells was investigated. We demonstrate that the mechanism responsible for the release of GP is ectodomain shedding mediated by cellular sheddases. Proteolytic cleavage taking place at amino-acid position D637 removes the transmembrane anchor and liberates complexes consisting of GP1 and truncated GP2 (GP(2delta)) subunits from the cell surface. We show that tumor necrosis factor alpha-converting enzyme (TACE), a member of the ADAM family of zinc-dependent metalloproteases, is involved in EBOV GP shedding. This finding shows for the first time that virus-encoded surface glycoproteins are substrates for ADAMs. Furthermore, we provide evidence that shed GP is present in significant amounts in the blood of virus-infected animals and that it may play an important role in the pathogenesis of infection by efficiently blocking the activity of virus-neutralizing antibodies.     

Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses, i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502–527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.     

Opisthorchis viverrini antigens up-regulates the expression of CD80 and MHC class II in JAWSII mouse dendritic cells and promotes IL-10 and TGF-β secretions

Dendritic cells (DCs) are antigen-presenting cells (APC) involved in the initiation of immune responses. Maturation of DCs is characterized by the high expression of major histocompatibility complex (MHC) class II and co-stimulatory clusters of differentiation (CD) 40, CD80, and CD86 molecules. Matured DCs are required for T cell differentiation and proliferation. However, the response of DCs to Opisthorchis viverrini antigens has not yet been understood. Therefore, this study sought to determine the expression of surface molecules of JAWSII mouse DCs stimulated by crude somatic (CS) and excretory-secretory (ES) antigens of O. viverrini. ES antigen significantly induced only mRNA expression of CD80 and MHC class II in JAWSII mouse DCs, while CS antigen promoted up-regulation of both mRNA and protein levels of CD80 and MHC class II, indicating relative maturation of JAWII mouse DCs. Moreover, the secreted cytokines from the co-cultures of O. viverrini antigens stimulated JAWSII DC with naïve CD4⁺ T cells was determined. Significantly increased levels of immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-β) were found. The up-regulation of these cytokines may indicate the response of regulatory T cells (Treg) to CS antigen-stimulated JAWSII DC. These findings may lead to a better understanding of the role that DCs play in O. viverrini infection.     

Ebola Virus Can Be Effectively Neutralized by Antibody Produced in Natural Human Infection

The activity of antibodies against filoviruses is poorly understood but has important consequences for vaccine design and passive prophylaxis. To investigate this activity, a panel of recombinant human monoclonal antibodies to Ebola virus antigens was isolated from phage display libraries constructed from RNA from donors who recovered from infection in the 1995 Ebola virus outbreak in Kikwit, Democratic Republic of Congo. Antibodies reactive with nucleoprotein (NP), envelope glycoprotein (GP), and secreted envelope glycoprotein (sGP) were characterized by immunofluorescence and radioimmunoprecipitation assays. Four antibodies reacting strongly with sGP and weakly with GP and two antibodies reacting with NP were not neutralizing. An antibody specific for GP neutralized Ebola virus to 50% at 0.4 microgram/ml as the recombinant Fab fragment and to 50% at 0.3 microgram/ml (90% at 2.6 microgram/ml) as the corresponding whole immunoglobulin G1 molecule. The studies indicate that neutralizing antibodies are produced in infection by Ebola virus although probably at a relatively low frequency. The neutralizing antibody may be useful in vaccine design and as a prophylactic agent against Ebola virus infection.     

High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-α in lung cancer

INTRODUCTION: Antigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-alpha induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-alpha within the microenvironment of primary lung carcinomas. RESULTS: Higher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-alpha-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry. CONCLUSION: These data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.     

Induction of humoral and CD8+ T cell responses are required for protection against lethal Ebola virus infection

Ebola virus (EBOV)-like particles (eVLP), composed of the EBOV glycoprotein and matrix viral protein (VP)40 with a lipid membrane, are a highly efficacious method of immunization against EBOV infection. The exact requirements for immunity against EBOV infection are poorly defined at this time. The goal of this work was to determine the requirements for EBOV immunity following eVLP vaccination. Vaccination of BALB/c or C57BL/6 mice with eVLPs in conjunction with QS-21 adjuvant resulted in mixed IgG subclass responses, a Th1-like memory cytokine response, and protection from lethal EBOV challenge. Further, this vaccination schedule led to the generation of both CD4(+) and CD8(+) IFN-gamma(+) T cells recognizing specific peptides within glycoprotein and VP40. The transfer of both serum and splenocytes, but not serum or splenocytes alone, from eVLP-vaccinated mice conferred protection against lethal EBOV infection in these studies. B cells were required for eVLP-mediated immunity to EBOV because B cell-deficient mice vaccinated with eVLPs were not protected from lethal EBOV challenge. We also found that CD8(+), but not CD4(+), T cells are absolutely required for eVLP-mediated protection against EBOV infection. Further, eVLP-induced protective mechanisms were perforin-independent, but IFN-gamma-dependent. Taken together, both EBOV-specific humoral and cytotoxic CD8(+) T cell responses are critical to mediate protection against filoviruses following eVLP vaccination.     

Characterization of monoclonal antibodies against waterfowl parvoviruses VP3 protein

Background

Ebola viruses (EBOVs) cause severe hemorrhagic fever with a high mortality rate. At present, there are no licensed vaccines or efficient therapies to combat EBOV infection. Previous studies have shown that both humoral and cellular immune responses are crucial for controlling Ebola infection. CD8⁺ T cells play an important role in mediating vaccine-induced protective immunity. The objective of this study was to identify H-2^d-specific T cell epitopes in EBOV glycoproteins (GPs).

Results

Computer-assisted algorithms were used to predict H-2^d-specific T cell epitopes in two species of EBOV (Sudan and Zaire) GP. The predicted peptides were synthesized and identified in BALB/c mice immunized with replication-deficient adenovirus vectors expressing the EBOV GP. Enzyme-linked immunospot assays and intracellular cytokine staining showed that the peptides RPHTPQFLF (Sudan EBOV), GPCAGDFAF and LYDRLASTV (Zaire EBOV) could stimulate splenoctyes in immunized mice to produce large amounts of interferon-gamma.

Conclusion

Three peptides within the GPs of two EBOV strains were identified as T cell epitopes. The identification of these epitopes should facilitate the evaluation of vaccines based on the Ebola virus glycoprotein in a BALB/c mouse model.     

The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains

Ebola virus (EBOV) infections are characterized by deficient T lymphocyte responses, T lymphocyte apoptosis, and lymphopenia in the absence of direct infection of T lymphocytes. In contrast, dendritic cells (DC) are infected but fail to mature appropriately, thereby impairing the T cell response. We investigated the contributions of EBOV proteins in modulating DC maturation by generating recombinant viruses expressing enhanced green fluorescent protein and carrying mutations affecting several potentially immunomodulating domains. They included envelope glycoprotein (GP) domains, as well as innate response antagonist domains (IRADs) previously identified in the VP24 and VP35 proteins. GP expressed by an unrelated vector, but not the wild-type EBOV, was found to strongly induce DC maturation, and infections with recombinant EBOV carrying mutations disabling GP functional domains did not restore DC maturation. In contrast, each of the viruses carrying mutations disabling any IRAD in VP35 induced a dramatic upregulation of DC maturation markers. This was dependent on infection, but not interaction with GP. Disabling of IRADs also resulted in up to a several hundredfold increase in secretion of cytokines and chemokines. Furthermore, these mutations induced formation of homotypic DC clusters, which represent close correlates of their maturation and presumably facilitate transfer of antigen from migratory DC to lymph node DC. Thus, an individual IRAD is insufficient to suppress DC maturation; rather, the suppression of DC maturation and the “immune paralysis” observed during EBOV infections results from a cooperative effect of two or more individual IRADs.     

Cathepsin B & L Are Not Required for Ebola Virus Replication

Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d''Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB^−/− and catL^−/− mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.     

Zaire Ebola virus entry into human dendritic cells is insensitive to cathepsin L inhibition

Cathepsins B and L contribute to Ebola virus (EBOV) entry into Vero cells and mouse embryonic fibroblasts. However, the role of cathepsins in EBOV‐infection of human dendritic cells (DCs), important targets of infection in vivo, remains undefined. Here, EBOV‐like particles containing a β‐lactamase–VP40 fusion reporter and Ebola virus were used to demonstrate the cathepsin dependence of EBOV entry into human monocyte‐derived DCs. However, while DC infection is blocked by cathepsin B inhibitor, it is insensitive to cathepsin L inhibitor. Furthermore, DCs pre‐treated for 48 h with TNFα were generally less susceptible to entry and infection by EBOV. This decrease in infection was associated with a decrease in cathepsin B activity. Thus, cathepsin L plays a minimal, if any, role in EBOV infection in human DCs. The inflammatory cytokine TNFα modulates cathepsin B activity and affects EBOV entry into and infection of human DCs.     

Coevolution of markers of innate and adaptive immunity in skin and peripheral blood of patients with erythema migrans

We used multiparameter flow cytometry to characterize leukocyte immunophenotypes and cytokines in skin and peripheral blood of patients with erythema migrans (EM). Dermal leukocytes and cytokines were assessed in fluids aspirated from epidermal suction blisters raised over EM lesions and skin of uninfected controls. Compared with corresponding peripheral blood, EM infiltrates were enriched for T cells, monocytes/macrophages, and dendritic cells (DCs), contained lower proportions of neutrophils, and were virtually devoid of B cells. Enhanced expression of CD14 and HLA-DR by lesional neutrophils and macrophages indicated that these innate effector cells were highly activated. Staining for CD45RO and CD27 revealed that lesional T lymphocytes were predominantly Ag-experienced cells; furthermore, a subset of circulating T cells also appeared to be neosensitized. Lesional DC subsets, CD11c(+) (monocytoid) and CD11c(-) (plasmacytoid), expressed activation/maturation surface markers. Patients with multiple EM lesions had greater symptom scores and higher serum levels of IFN-alpha, TNF-alpha, and IL-2 than patients with solitary EM. IL-6 and IFN-gamma were the predominant cytokines in EM lesions; however, greater levels of both mediators were detected in blister fluids from patients with isolated EM. Circulating monocytes displayed significant increases in surface expression of Toll-like receptor (TLR)1 and TLR2, while CD11c(+) DCs showed increased expression of TLR2 and TLR4; lesional macrophages and CD11c(+) and CD11c(-) DCs exhibited increases in expression of all three TLRs. These results demonstrate that Borrelia burgdorferi triggers innate and adaptive responses during early Lyme disease and emphasize the interdependence of these two arms of the immune response in the efforts of the host to contain spirochetal infection.     

The Myeloid LSECtin Is a DAP12-Coupled Receptor That Is Crucial for Inflammatory Response Induced by Ebola Virus Glycoprotein

Fatal Ebola virus infection is characterized by a systemic inflammatory response similar to septic shock. Ebola glycoprotein (GP) is involved in this process through activating dendritic cells (DCs) and macrophages. However, the mechanism is unclear. Here, we showed that LSECtin (also known as CLEC4G) plays an important role in GP-mediated inflammatory responses in human DCs. Anti-LSECtin mAb engagement induced TNF-α and IL-6 production in DCs, whereas silencing of LSECtin abrogated this effect. Intriguingly, as a pathogen-derived ligand, Ebola GP could trigger TNF-α and IL-6 release by DCs through LSECtin. Mechanistic investigations revealed that LSECtin initiated signaling via association with a 12-kDa DNAX-activating protein (DAP12) and induced Syk activation. Mutation of key tyrosines in the DAP12 immunoreceptor tyrosine-based activation motif abrogated LSECtin-mediated signaling. Furthermore, Syk inhibitors significantly reduced the GP-triggered cytokine production in DCs. Therefore, our results demonstrate that LSECtin is required for the GP-induced inflammatory response, providing new insights into the EBOV-mediated inflammatory response.     

Highly pathogenic avian influenza H5N1 virus induces cytokine dysregulation with suppressed maturation of chicken monocyte‐derived dendritic cells

One of the major causes of death in highly pathogenic avian influenza virus (HPAIV) infection in chickens is acute induction of pro‐inflammatory cytokines (cytokine storm), which leads to severe pathology and acute mortality. DCs and respiratory tract macrophages are the major antigen presenting cells that are exposed to mucosal pathogens. We hypothesized that chicken DCs are a major target for induction of cytokine dysregulation by H5N1 HPAIV. It was found that infection of chicken peripheral blood monocyte‐derived dendritic cells (chMoDCs) with H5N1 HPAIV produces high titers of progeny virus with more rounding and cytotoxicity than with H9N2 LPAIV. Expression of maturation markers (CD40, CD80 and CD83) was weaker in both H5N1 and H9N2 groups than in a LPS control group. INF‐α, ‐β and ‐γ were significantly upregulated in the H5N1 group. Pro‐inflammatory cytokines (IL‐1β, TNF‐α and IL‐18) were highly upregulated in early mid (IL‐1), and late (IL‐6) phases of H5N1 virus infection. IL‐8 (CXCLi2) mRNA expression was significantly stronger in the H5N1 group from 6 hr of infection. TLR3, 7, 15 and 21 were upregulated 24 hr after infection by H5N1 virus compared with H9N2 virus, with maximum expression of TLR 3 mRNA. Similarly, greater H5N1 virus‐induced apoptotic cell death and cytotoxicity, as measured by terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling and lactate dehydrogenase assays, respectively, were found. Thus, both H5N1 and H9N2 viruses evade the host immune system by inducing impairment of chMoDCs maturation and enhancing cytokine dysregulation in H5N1 HPAIV‐infected cells.