Jiří Salát

CD8 + T-cells mediate immunopathology in tick-borne encephalitis

Epidemics of tick-borne encephalitis involving thousands of humans occur annually in the forested regions of Europe and Asia. Despite the importance of this disease, the underlying basis for the development of encephalitis remains undefined. Here, we prove the key role of CD8(+) T-cells in the immunopathology of tick-borne encephalitis, as demonstrated by prolonged survival of SCID or CD8(-/-) mice, following infection, when compared with immunocompetent mice or mice with adoptively transferred CD8(+) T-cells. The results imply that tick-borne encephalitis is an immunopathological disease and that the inflammatory reaction significantly contributes to the fatal outcome of the infection.

Breakdown of the blood-brain barrier during tick-borne encephalitis in mice is not dependent on CD8+ T-cells.

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. The disease is characterized by fever and debilitating encephalitis that can progress to chronic illness or fatal infection. In this study, changes in permeability of the blood-brain barrier (BBB) in two susceptible animal models (BALB/c, and C57Bl/6 mice) infected with TBE virus were investigated at various days after infection by measuring fluorescence in brain homogenates after intraperitoneal injection of sodium fluorescein, a compound that is normally excluded from the central nervous system. We demonstrate here that TBE virus infection, in addition to causing fatal encephalitis in mice, induces considerable breakdown of the BBB. The permeability of the BBB increased at later stages of TBE infection when high virus load was present in the brain (i.e., BBB breakdown was not necessary for TBE virus entry into the brain), and at the onset of the first severe clinical symptoms of the disease, which included neurological signs associated with sharp declines in body weight and temperature. The increased BBB permeability was in association with dramatic upregulation of proinflammatory cytokine/chemokine mRNA expression in the brain. Breakdown of the BBB was also observed in mice deficient in CD8+ T-cells, indicating that these cells are not necessary for the increase in BBB permeability that occurs during TBE. These novel findings are highly relevant to the development of future therapies designed to control this important human infectious disease.

Crystal structure and functional characterization of an immunomodulatory salivary cystatin from the soft tick Ornithodoros moubata

The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help to overcome the hosts defence during host-parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken in the present study. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and was found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 A (1 A=0.1 nm) and was used to describe the structure-inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the pro-inflammatory cytokines tumour necrosis factor alpha and interleukin-12, and proliferation of antigen-specific CD4+ T-cells. This suggests that OmC2 may suppress the hosts adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.

Mice with different susceptibility to tick-borne encephalitis virus infection show selective neutralizing antibody response and inflammatory reaction in the central nervous system

BackgroundThe clinical course of tick-borne encephalitis (TBE), a disease caused by TBE virus, ranges from asymptomatic or mild influenza-like infection to severe debilitating encephalitis or encephalomyelitis. Despite the medical importance of this disease, some crucial steps in the development of encephalitis remain poorly understood. In particular, the basis of the disease severity is largely unknown.MethodsTBE virus growth, neutralizing antibody response, key cytokine and chemokine mRNA production and changes in mRNA levels of cell surface markers of immunocompetent cells in brain were measured in mice with different susceptibilities to TBE virus infection.ResultsAn animal model of TBE based on BALB/c-c-STS/A (CcS/Dem) recombinant congenic mouse strains showing different severities of the infection in relation to the host genetic background was developed. After subcutaneous inoculation of TBE virus, BALB/c mice showed medium susceptibility to the infection, STS mice were resistant, and CcS-11 mice were highly susceptible. The resistant STS mice showed lower and delayed viremia, lower virus production in the brain and low cytokine/chemokine mRNA production, but had a strong neutralizing antibody response. The most sensitive strain (CcS-11) failed in production of neutralizing antibodies, but exhibited strong cytokine/chemokine mRNA production in the brain. After intracerebral inoculation, all mouse strains were sensitive to the infection and had similar virus production in the brain, but STS mice survived significantly longer than CcS-11 mice. These two strains also differed in the expression of key cytokines/chemokines, particularly interferon gamma-induced protein 10 (IP-10/CXCL10) and monocyte chemotactic protein-1 (MCP-1/CCL2) in the brain.ConclusionsOur data indicate that the genetic control is an important factor influencing the clinical course of TBE. High neutralizing antibody response might be crucial for preventing host fatality, but high expression of various cytokines/chemokines during TBE can mediate immunopathology and be associated with more severe course of the infection and increased fatality.

Nucleoside Inhibitors of Tick-Borne Encephalitis Virus

ABSTRACT Tick-borne encephalitis virus (TBEV) is a leading cause of human neuroinfections in Europe and Northeast Asia. There are no antiviral therapies for treating TBEV infection. A series of nucleoside analogues was tested for the ability to inhibit the replication of TBEV in porcine kidney cells and human neuroblastoma cells. The interactions of three nucleoside analogues with viral polymerase were simulated using advanced computational methods. The nucleoside analogues 7-deaza-2′-C-methyladenosine (7-deaza-2′-CMA), 2′-C-methyladenosine (2′-CMA), and 2′-C-methylcytidine (2′-CMC) inhibited TBEV replication. These compounds showed dose-dependent inhibition of TBEV-induced cytopathic effects, TBEV replication (50% effective concentrations [EC50]of 5.1 ± 0.4 μM for 7-deaza-2′-CMA, 7.1 ± 1.2 μM for 2′-CMA, and 14.2 ± 1.9 μM for 2′-CMC) and viral antigen production. Notably, 2′-CMC was relatively cytotoxic to porcine kidney cells (50% cytotoxic concentration [CC50] of ∼50 μM). The anti-TBEV effect of 2′-CMA in cell culture diminished gradually after day 3 posttreatment. 7-Deaza-2′-CMA showed no detectable cellular toxicity (CC50 > 50 μM), and the antiviral effect in culture was stable for >6 days posttreatment. Computational molecular analyses revealed that compared to the other two compounds, 7-deaza-2′-CMA formed a large cluster near the active site of the TBEV polymerase. High antiviral activity and low cytotoxicity suggest that 7-deaza-2′-CMA is a promising candidate for further investigation as a potential therapeutic agent in treating TBEV infection.

Infection and injury of human astrocytes by tick-borne encephalitis virus

Tick-borne encephalitis (TBE), a disease caused by tick-borne encephalitis virus (TBEV), represents the most important flaviviral neural infection in Europe and north-eastern Asia. In the central nervous system (CNS), neurons are the primary target for TBEV infection; however, infection of non-neuronal CNS cells, such as astrocytes, is not well understood. In this study, we investigated the interaction between TBEV and primary human astrocytes. We report for the first time, to the best of our knowledge, that primary human astrocytes are sensitive to TBEV infection, although the infection did not affect their viability. The infection induced a marked increase in the expression of glial fibrillary acidic protein, a marker of astrocyte activation. In addition, expression of matrix metalloproteinase 9 and several key pro-inflammatory cytokines/chemokines (e.g. tumour necrosis factor α, interferon α, interleukin (IL)-1β, IL-6, IL-8, interferon γ-induced protein 10, macrophage inflammatory protein, but not monocyte chemotactic protein 1) was upregulated. Moreover, we present a detailed description of morphological changes in TBEV-infected cells, as investigated using three-dimensional electron tomography. Several novel ultrastructural changes were observed, including the formation of unique tubule-like structures of 17.9 ±0.15 nm diameter with associated viral particles and/or virus-induced vesicles and located in the rough endoplasmic reticulum of the TBEV-infected cells. This is the first demonstration that TBEV infection activates primary human astrocytes. The infected astrocytes might be a potential source of pro-inflammatory cytokines in the TBEV-infected brain, and might contribute to the TBEV-induced neurotoxicity and blood-brain barrier breakdown that occurs during TBE. The neuropathological significance of our observations is also discussed.

CD8+ T lymphocytes protect SCID mice against Encephalitozoon cuniculi infection

Microsporidia are obligate intracellular parasites that cause opportunistic infections in immunocompromised patients. The role of two main T cell subsets in anti-microsporidial immunity has been studied using an Encephalitozoon cuniculi-severe combined immunodeficient (SCID) mouse model. Whereas SCID mice reconstituted with CD4+ T lymphocyte-depleted naive BALB/c splenocytes resolved the infection, adoptive transfer of CD8+ T cell-depleted splenocytes failed to protect the animals against a lethal E. cuniculi infection. Splenocytes from E. cuniculi-immune mice specifically killed syngeneic infected macrophages in a short-term 51Cr-release assay. These results suggest the crucial role of cytotoxic T lymphocytes in the protection against E. cuniculi infection.

Analysis of serum levels of cytokines, chemokines, growth factors, and monoamine neurotransmitters in patients with tick‐borne encephalitis: Identification of novel inflammatory markers with implications for pathogenesis

Tick‐borne encephalitis (TBE) is a leading human neuroinfection in Europe and northeastern Asia. However, the pathophysiology of TBE is not understood completely. This study sought to determine the specific serum mediators that are associated with acute TBE. The levels of 30 cytokines, chemokines, and growth factors were measured in serum samples from 87 patients with clinically and serologically confirmed acute TBE and from 32 control subjects using the Cytokine Human Magnetic 30‐Plex Panel for the Luminex platform. Serum levels of the monoamine neurotransmitters serotonin, dopamine, and noradrenaline were measured via enzyme‐linked immunosorbent assay. TBE virus infection elicited increased levels of the pro‐inflammatory cytokines interleukin (IL)‐6, IL‐8, and IL‐12. TBE patients had higher IL‐12:IL‐4 and IL‐12:IL‐10 ratios than control patients, reflecting the global pro‐inflammatory cytokine balance. Serum levels of the monoamine neurotransmitters serotonin, dopamine, and noradrenaline were significantly lower in TBE patients than in the control group. Most interestingly, increased levels of hepatocyte growth factor and vascular endothelial growth factor were observed in TBE patients; these proteins may be novel and mechanistically important inflammatory biomarkers of TBE. J. Med. Virol. 87:885–892, 2015.

Anti-tumour necrosis factor-alpha activity in Ixodes ricinus saliva.

Tumour necrosis factor‐α (TNF‐α) is one of the most prominent inflammatory mediators playing a central role in starting off the inflammatory reactions of the innate immune system. We identified a TNF‐α‐inhibitory activity in the saliva and salivary gland extract (SGE) from partially fed Ixodes ricinus ticks. Using mouse and human TNF‐α specific ELISA, we showed that tick saliva or SGE markedly reduced the level of detectable cytokine. Both saliva and SGE inhibited the cytotoxic effect of TNF‐α in a bioassay. Elimination of the TNF‐α‐inhibitory activity in SGE by trypsin digestion demonstrated that the anti‐TNF‐α factor is a protein. Fast protein liquid chromatography fractionation of SGE showed one peak of TNF‐α‐inhibitory activity corresponding to a protein with estimated molecular mass 23 kDa. The likely mechanism of the inhibitory effect is a direct binding of the cytokine. The TNF‐α‐inhibitory molecule seems to play an important role in the anti‐inflammatory effect of tick saliva at the tick feeding site, providing a gateway to the host for tick‐borne pathogens.

Antibodies enhance the protective effect of CD4+ T lymphocytes in SCID mice perorally infected with Encephalitozoon cuniculi

The role of antibodies in the immune response to microsporidiosis was studied using a novel anti‐exospore monoclonal antibody (MAb) P5/H1, which recognizes surface antigens of Encephalitozoon cuniculi. The effect of the MAb on microsporidial infection in vivo was to prolong the survival of previously CD4+ reconstituted, perorally infected and intraperitoneally MAb‐treated SCID mice. The MAb decreased the numbers of E. cuniculi spores in peritoneal smears obtained post mortem. These results suggest a possible role for antibodies in protection against perorally acquired E. cuniculi infection.