Christiane Schnabel - Academia.edu (original) (raw)

Papers by Christiane Schnabel

Research paper thumbnail of Total anti-EHV-1 gC and IgG isotype responses in serum from horses after infection with the EHV-1 strain Ab4 or its deletion mutant Ab4ΔORF1/71

<p>EHV-1 naïve horses (n = 5 per group) were infected with the EHV-1 strain Ab4 or Ab4ΔORF1... more <p>EHV-1 naïve horses (n = 5 per group) were infected with the EHV-1 strain Ab4 or Ab4ΔORF1/71. Non-infected horses were kept as controls. The arrow marks the time of infection on d0. Serum antibodies were measured by an EHV-1 Multiplex assay. Antibody values are expressed as median fluorescence intensities (MFI) for (A) total Ig, (B) IgG1, (C) IgG1/3, and (D) IgG4/7. All graphs show means and standard errors by group over time. The dotted vertical lines mark d8 and d12pi to illustrate the time of the onset of the response for total Ig and the different IgG isotypes. Significant differences between the groups are shown: a = Ab4 vs. controls, b = Ab4ΔORF1/71 vs. controls, and c = Ab4 vs. Ab4ΔORF1/71.</p

Research paper thumbnail of Comprehensive Flow Cytometric Characterization of Bronchoalveolar Lavage Cells Indicates Comparable Phenotypes Between Asthmatic and Healthy Horses But Functional Lymphocyte Differences

Frontiers in Immunology

Equine asthma (EA) is a highly relevant disease, estimated to affect up to 20% of all horses, and... more Equine asthma (EA) is a highly relevant disease, estimated to affect up to 20% of all horses, and compares to human asthma. The pathogenesis of EA is most likely immune-mediated, yet incompletely understood. To study the immune response in the affected lower airways, mixed leukocytes were acquired through bronchoalveolar lavage (BAL) and the cell populations were analyzed on a single-cell basis by flow cytometry (FC). Samples of 38 horses grouped as respiratory healthy or affected by mild to moderate (mEA) or severe EA (sEA) according to their history, clinical signs, and BAL cytology were analyzed. Using FC, BAL cells and PBMC were comprehensively characterized by cell surface markers ex vivo. An increased percentage of DH24A+ polymorphonuclear cells, and decreased percentages of CD14+ macrophages were detected in BAL from horses with sEA compared to healthy horses or horses with mEA, while lymphocyte proportions were similar between all groups. Independently of EA, macrophages in ...

Research paper thumbnail of CD154 Expression Indicates T Cell Activation Following Tetanus Toxoid Vaccination of Horses

Frontiers in Immunology

Despite the relevance of adaptive immunity against equine pathogens antigen-specific T cell respo... more Despite the relevance of adaptive immunity against equine pathogens antigen-specific T cell responses of horses are not well characterized and the lack of insight into T cell responses hampers the understanding of the pathogeneses of important diseases. In this study we used tetanus toxoid (TT) as a well-defined antigen to characterize antigen-reactive T cells. Six healthy adult horses received a routine booster against tetanus with an immune stimulating complex (ISCOM)-based vaccine and were followed for 28 days. TT-specific serum antibodies were quantified by ELISA and increased in all horses by day 7 after vaccination. CD154 is an established indicator of antigen-reactive T helper cells in other species, but has not been characterized in horses. CD154 detection in equine PBMC by an anti-human CD154 antibody (clone 5C8) was confirmed by Western blots and then applied for flow cytometry. As a common indicator of equine T cell activation, cytokine induction was studied in parallel. ...

Research paper thumbnail of New mAbs facilitate quantification of secreted equine TNF-α and flow cytometric analysis in monocytes and T cells

Veterinary Immunology and Immunopathology, 2021

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, that is involved in acute inflammation... more Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, that is involved in acute inflammation and is employed as a biomarker of inflammatory diseases in several species for which reliable quantification is available. We aimed to develop suitable tools to quantify TNF-α in equine samples. We generated two new mAbs against equine TNF-α (clones 48 and 292), evaluated their specificity for this cytokine, and confirmed detection of native TNF-α in stimulated equine PBMC. The TNF-α mAbs were paired in a fluorescent bead-based assay for quantification of equine TNF-α. The TNF-α assay had a wide quantification range of 12 pg/mL - 38.4 ng/mL. In addition, TNF-α mAb 48 was used for a detailed analysis of TNF-α production in PBMC by intracellular staining and flow cytometry. TNF-α was expressed by CD14+ monocytes after LPS stimulation and by monocytes and lymphocytes after polyclonal stimulation with PMA and ionomycin in vitro. TNF-α expressing lymphocytes consisted mainly of CD4+ T cells. CD8+ T cells and other lymphocytes also expressed TNF-α. The new mAbs evaluated here for soluble and intracellular TNF-α will enable the detailed analysis of this important pro-inflammatory cytokine during equine immune responses and inflammatory diseases of the horse.

Research paper thumbnail of Additional file 1: of Deletion of the ORF2 gene of the neuropathogenic equine herpesvirus type 1 strain Ab4 reduces virulence while maintaining strong immunogenicity

Figure S1. Cytokines in serum after infection with EHV-1 strain Ab4 or its deletion mutant Ab4ΔOR... more Figure S1. Cytokines in serum after infection with EHV-1 strain Ab4 or its deletion mutant Ab4ΔORF2. Horses (n = 8/group) were infected with one of two EHV-1 strains (Ab4) or Ab4ΔORF2 at day 0 or kept as uninfected controls. Serum was sampled at several times before (days − 8, − 1) and after (day 1- day 24) infection, cytokines and the inflammatory marker sCD14 were evaluated with fluorescent bead-based assays. Mean and standard errors for IFN-α (A), sCD14 (B), and IFN-γ (C) in the serum are displayed. Significant differences between groups are marked: a = Ab4 vs. control, b = Ab4ΔORF2 vs. control, c = Ab4 vs. Ab4ΔORF2. (JPG 332 kb)

Research paper thumbnail of Identification of Disease-Associated Cryptococcal Proteins Reactive With Serum IgG From Cryptococcal Meningitis Patients

Frontiers in Immunology, 2021

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment... more Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera fromC. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung funga...

Research paper thumbnail of RESEARCH ARTICLE Open Access Immune

response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent

Research paper thumbnail of Peripheral blood basophils are the main source for early interleukin-4 secretion upon in vitro stimulation with Culicoides allergen in allergic horses

PLOS ONE, 2021

Interleukin-4 (IL-4) is a key cytokine secreted by type 2 T helper (Th2) cells that orchestrates ... more Interleukin-4 (IL-4) is a key cytokine secreted by type 2 T helper (Th2) cells that orchestrates immune responses during allergic reactions. Human and mouse studies additionally suggest that basophils have a unique role in the regulation of allergic diseases by providing initial IL-4 to drive T cell development towards the Th2 phenotype. EquineCulicoideshypersensitivity (CH) is a seasonal immunoglobulin E (IgE)-mediated allergic dermatitis in horses in response to salivary allergens fromCulicoides(Cul) midges. Here, we analyzed IL-4 production in peripheral blood mononuclear cells (PBMC) of CH affected (n = 8) and healthy horses (n = 8) living together in an environment with naturalCulexposure. DuringCulexposure when allergic horses had clinical allergy, IL-4 secretion from PBMC after stimulation withCulextract was similar between healthy and CH affected horses. In contrast, allergic horses had higher IL-4 secretion from PBMC than healthy horses during months without allergen exposu...

Research paper thumbnail of An Equine Herpesvirus Type 1 (EHV-1) Ab4 Open Reading Frame 2 Deletion Mutant Provides Immunity and Protection from EHV-1 Infection and Disease

Journal of Virology, 2019

Nasal equine herpesvirus type 1 (EHV-1) shedding is essential for virus transmission during outbr... more Nasal equine herpesvirus type 1 (EHV-1) shedding is essential for virus transmission during outbreaks. Cell-associated viremia is a prerequisite for the most severe disease outcomes, abortion and equine herpesvirus myeloencephalopathy (EHM). Thus, protection from viremia is considered essential for preventing EHM. Ab4ΔORF2 vaccination prevented EHV-1 challenge virus replication in the upper respiratory tract in fully protected horses. Consequently, these neither shed virus nor developed cell-associated viremia. Protection from virus shedding and viremia during challenge infection in combination with reduced virulence at the time of vaccination emphasizes ORF2 deletion as a promising modification for generating an improved EHV-1 vaccine. During this challenge infection, full protection was linked to preexisting local and systemic EHV-1-specific antibodies combined with rapidly increasing intranasal IgG4/7 antibodies and lack of nasal type I interferon and chemokine induction. These h...

Research paper thumbnail of Deletion of the ORF2 gene of the neuropathogenic equine herpesvirus type 1 strain Ab4 reduces virulence while maintaining strong immunogenicity

BMC Veterinary Research, 2018

Background: Equine herpesvirus type 1 (EHV-1) induces respiratory infection, abortion, and neurol... more Background: Equine herpesvirus type 1 (EHV-1) induces respiratory infection, abortion, and neurologic disease with significant impact. Virulence factors contributing to infection and immune evasion are of particular interest. A potential virulence factor of the neuropathogenic EHV-1 strain Ab4 is ORF2. This study on 24 Icelandic horses, 2 to 4 years of age, describes the infection with EHV-1 Ab4, or its deletion mutant devoid of ORF2 (Ab4ΔORF2) compared to non-infected controls (each group n = 8). The horses' clinical presentation, virus shedding, viremia, antibody and cellular immune responses were monitored over 260 days after experimental infection. Results: Infection with Ab4ΔORF2 reduced fever and minimized nasal virus shedding after infection compared to the parent virus strain Ab4, while Ab4ΔORF2 established viremia similar to Ab4. Concurrently with virus shedding, intranasal cytokine and interferon α (IFN-α) production increased in the Ab4 group, while horses infected with Ab4ΔORF2 expressed less IFN-α. The antibody response to EHV-1 was evaluated by a bead-based multiplex assay and was similar in both infected groups, Ab4 and Ab4ΔORF2. EHV-1 specific immunoglobulin (Ig) G1 was induced 8 days after infection (d8 pi) with a peak on d10-12 pi. EHV-1 specific IgG4/7 increased starting on d10 pi, and remained elevated in serum until the end of the study. The intranasal antibody response to EHV-1 was dominated by the same IgG isotypes and remained elevated in both infected groups until d130 pi. In contrast to the distinct antibody response, no induction of EHV-1 specific T-cells was detectable by flow cytometry after ex vivo re-stimulation of peripheral blood mononuclear cells (PBMC) with EHV-1 in any group. The cellular immune response was characterized by increased secretion of IFN-γ and interleukin10 in response to ex vivo re-stimulation of PBMC with EHV-1. This response was present during the time of viremia (d5-10 pi) and was similar in both infected groups, Ab4 and Ab4ΔORF2. Conclusions: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa. In contrast, ORF2 does not influence viremia. The immunogenicity of the Ab4ΔORF2 and parent Ab4 viruses are identical.

Research paper thumbnail of C-C motif chemokine ligand (CCL) production in equine peripheral blood mononuclear cells identified by newly generated monoclonal antibodies

Veterinary Immunology and Immunopathology, 2018

Chemokines are soluble molecules directing immune cell trafficking and homing, mediating inflamma... more Chemokines are soluble molecules directing immune cell trafficking and homing, mediating inflammation, and initiating immune responses to infection. In horses, the analysis of chemokines has been limited by the lack of specific antibodies. We generated mAbs specific for the equine CC motif chemokine ligands (CCL) CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES) and CCL11 (eotaxin) using hybridoma technology. Antibody specificity was confirmed by intracellular staining of Chinese Hamster Ovary cells transfected with expression vectors encoding for CCL2, CCL3, CCL5, or CCL11. Transfectants were stained with the anti-CCL mAbs. Flow cytometric analysis confirmed the specificity of the different mAbs for the respective chemokine. In addition, equine PBMC were stained after isolation, culture in medium, or stimulation with LPS, or PMA and ionomycin. CCL2 was detected in few cluster of differentiation (CD)14 + monocytes in PBMC stimulated with PMA and ionomycin for 2 h. CCL3 was produced by CD14 + monocytes after 4-6 h culture in medium. After stimulation with PMA and ionomycin for 12-24 h, CCL3 was also expressed in lymphocytes, mainly in CD4 + T cells. Stimulation with LPS reduced the percentage of CCL3 + monocytes in PBMC. CCL5 was detected in PBMC ex vivo in CD4 + and CD8 + T cells. Culture of PBMC for longer than 6 h or stimulation with PMA and ionomycin reduced the percentage of CCL5 + cells. CCL11 was produced by CD4 + T cells in PBMC after stimulation with PMA and ionomycin for 4-24 h. After LPS stimulation of PBMC, CCL2, CCL5, and CCL11 production were comparable to culture in medium alone. ELISAs for each of the four chemokines were developed using pairs of anti-equine CCL mAbs. Supernatants from PMA and ionomycin stimulated PBMC contained detectable amounts of CCL2, CCL3 and CCL5, while CCL11 secretion could be stimulated from equine tracheal epithelial cells in response to IL-4. The newly generated mAbs for equine CCL chemokines facilitate the quantitative analysis of intracellular chemokine production by flow cytometry and soluble chemokines by ELISA. The CCL mAbs are valuable tools to improve the evaluation of innate immune responses in horses.

Research paper thumbnail of CXCL10 production in equine monocytes is stimulated by interferon-gamma

Veterinary Immunology and Immunopathology, 2018

C-X-C motif ligand 10 (CXCL10) is a pro-inflammatory chemokine and has been extensively evaluated... more C-X-C motif ligand 10 (CXCL10) is a pro-inflammatory chemokine and has been extensively evaluated in people and mice. In horses, CXCL10 and its involvement in host immunity has rarely been analyzed due to the lack of specific antibodies. We generated a mAb specific for the equine chemokine CXCL10 using hybridoma technology. Antibody specificity was confirmed by intracellular staining and flow cytometric analysis of Chinese Hamster Ovary (CHO) cells expressing equine rCXCL10, while CHO cells expressing equine rCXCL9 were not detected. Native CXCL10 expression in PBMC from horses of different age groups was analyzed by flow cytometry after in vitro stimulation. CXCL10 expressing PBMC were characterized by triple staining of CXCL10 combined with cell-surface markers. Stimulation with IFN-γ for 5 h similarly induced CXCL10 production in cluster of differentiation (CD) 14 + CD16 − MHCII high monocytes of adult horses and weanlings. The newly generated mAb enables the quantitative intracellular analysis of CXCL10 by flow cytometry and provides a new valuable tool to improve the evaluation of inflammatory responses in horses.

Research paper thumbnail of The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses

PLOS ONE, 2018

The equine herpesvirus type 1 (EHV-1) ORF1 and ORF71 genes have immune modulatory effects in vitr... more The equine herpesvirus type 1 (EHV-1) ORF1 and ORF71 genes have immune modulatory effects in vitro. Experimental infection of horses using virus mutants with multiple deletions including ORF1 and ORF71 showed promise as vaccine candidates against EHV-1. Here, the combined effects of ORF1 and ORF71 deletions from the neuropathogenic EHV-1 strain Ab4 on clinical disease and host immune response were further explored. Three groups of EHV-1 naïve horses were experimentally infected with the ORF1/71 gene deletion mutant (Ab4ΔORF1/71), the parent Ab4 strain, or remained uninfected. In comparison to Ab4, horses infected with Ab4ΔORF1/71 did not show the initial high fever peak characteristic of EHV-1 infection. Ab4ΔORF1/71 infection had reduced nasal shedding (1/5 vs. 5/5) and, simultaneously, decreased intranasal interferon (IFN)-α, interleukin (IL)-10 and soluble CD14 secretion. However, Ab4 and Ab4ΔORF1/71 infection resulted in comparable viremia, suggesting these genes do not regulate the infection of the mononuclear cells and subsequent viremia. Intranasal and serum anti-EHV-1 antibodies to Ab4ΔORF1/71 developed slightly slower than those to Ab4. However, beyond day 12 post infection (d12pi) serum antibodies in both virusinfected groups were similar and remained increased until the end of the study (d114pi). EHV-1 immunoglobulin (Ig) G isotype responses were dominated by short-lasting IgG1 and long-lasting IgG4/7 antibodies. The IgG4/7 response closely resembled the total EHV-1 specific antibody response. Ex vivo re-stimulation of PBMC with Ab4 resulted in IFN-γ and IL-10 secretion by cells from both infected groups within two weeks pi. Flow cytometric analysis showed that IFN-γ producing EHV-1-specific T-cells were mainly CD8 + /IFN-γ + and detectable from d32pi on. Peripheral blood IFN-γ + T-cell percentages were similar in both infected groups, albeit at low frequency (~0.1%). In summary, the Ab4ΔORF1/71 gene deletion mutant is less virulent but induced antibody responses and cellular immunity similar to the parent Ab4 strain.

Research paper thumbnail of Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay

Veterinary Immunology and Immunopathology, 2017

Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (I... more Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse. Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observed. The new IgA mAb 84-1 was used in combination with the previously characterized anti-equine IgA mAb BVS2 for the development and validation of a fluorescent bead-based assay to quantify total IgA in equine serum and various secretions. The IgA assay's linear detection ranged from 64 pg/ml to 1000 ng/ml. For the quantification of IgA in serum or in secretions an IgA standard was purified from serum or nasal wash fluid (secretory IgA), respectively. The different standards were needed for accurate IgA quantification in the respective samples taking the different signal intensities of monomeric and dimeric IgA on the florescent bead-based assay into account. IgA was quantified by the bead-based assay established here in different equine samples of healthy adult individuals. In serum the median total IgA was 0.45 mg/ml for Thoroughbred horses (TB, n = 10) and 1.16 mg/ ml in Icelandic horses (ICH, n = 12). In nasopharyngeal secretions of TB (n = 7) 0.13 mg/ml median total IgA was measured, and 0.25 mg/ml for ICH (n = 12). Saliva of ICH (n = 6) contained a median of 0.15 mg/ml, colostrum of Warmbloods (n = 8) a median of 1.89 mg/ml IgA. Compared to IgG1 and IgG4/7 quantified in the same samples, IgA appeared as the major immunoglobulin isotype in nasopharyngeal secretions and saliva while it is a minor isotype in serum and colostrum. The newly developed monoclonal antibodies against equine IgA and the resulting bead-based assay for quantification of total IgA can notably improve the evaluation of mucosal immunity in horses.

Research paper thumbnail of Influences of age and sex on leukocytes of healthy horses and their ex vivo cytokine release

Veterinary immunology and immunopathology, Jan 6, 2015

Leukocytes and their functional capacities are used extensively as biomarkers in immunological re... more Leukocytes and their functional capacities are used extensively as biomarkers in immunological research. Commonly employed indicators concerning leukocytes are as follows: number, composition in blood, response to discrete stimuli, cytokine release, and morphometric characteristics. In order to employ leukocytes as biomarkers for disease and therapeutic monitoring, physiological variations and influencing factors on the parameters measured have to be considered. The aim of this report was to describe the ranges of selected leukocyte parameters in a sample of healthy horses and to analyse whether age, sex, breed, and sampling time point (time of day) influence peripheral blood leukocyte composition, cell morphology and release of cytokines ex vivo. Flow cytometric comparative characterisation of cell size and complexity in 24 healthy horses revealed significant variance. Similarly, basal release of selected cytokines by blood mononuclear cells also showed high variability [TNFα (65-1...

Research paper thumbnail of Evaluation of the reactivity of commercially available monoclonal antibodies with equine cytokines

Veterinary Immunology and Immunopathology, 2013

Research paper thumbnail of Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent

BMC veterinary research, Jan 23, 2015

Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. ... more Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown. In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte num...

Research paper thumbnail of Total anti-EHV-1 gC and IgG isotype responses in serum from horses after infection with the EHV-1 strain Ab4 or its deletion mutant Ab4ΔORF1/71

<p>EHV-1 naïve horses (n = 5 per group) were infected with the EHV-1 strain Ab4 or Ab4ΔORF1... more <p>EHV-1 naïve horses (n = 5 per group) were infected with the EHV-1 strain Ab4 or Ab4ΔORF1/71. Non-infected horses were kept as controls. The arrow marks the time of infection on d0. Serum antibodies were measured by an EHV-1 Multiplex assay. Antibody values are expressed as median fluorescence intensities (MFI) for (A) total Ig, (B) IgG1, (C) IgG1/3, and (D) IgG4/7. All graphs show means and standard errors by group over time. The dotted vertical lines mark d8 and d12pi to illustrate the time of the onset of the response for total Ig and the different IgG isotypes. Significant differences between the groups are shown: a = Ab4 vs. controls, b = Ab4ΔORF1/71 vs. controls, and c = Ab4 vs. Ab4ΔORF1/71.</p

Research paper thumbnail of Comprehensive Flow Cytometric Characterization of Bronchoalveolar Lavage Cells Indicates Comparable Phenotypes Between Asthmatic and Healthy Horses But Functional Lymphocyte Differences

Frontiers in Immunology

Equine asthma (EA) is a highly relevant disease, estimated to affect up to 20% of all horses, and... more Equine asthma (EA) is a highly relevant disease, estimated to affect up to 20% of all horses, and compares to human asthma. The pathogenesis of EA is most likely immune-mediated, yet incompletely understood. To study the immune response in the affected lower airways, mixed leukocytes were acquired through bronchoalveolar lavage (BAL) and the cell populations were analyzed on a single-cell basis by flow cytometry (FC). Samples of 38 horses grouped as respiratory healthy or affected by mild to moderate (mEA) or severe EA (sEA) according to their history, clinical signs, and BAL cytology were analyzed. Using FC, BAL cells and PBMC were comprehensively characterized by cell surface markers ex vivo. An increased percentage of DH24A+ polymorphonuclear cells, and decreased percentages of CD14+ macrophages were detected in BAL from horses with sEA compared to healthy horses or horses with mEA, while lymphocyte proportions were similar between all groups. Independently of EA, macrophages in ...

Research paper thumbnail of CD154 Expression Indicates T Cell Activation Following Tetanus Toxoid Vaccination of Horses

Frontiers in Immunology

Despite the relevance of adaptive immunity against equine pathogens antigen-specific T cell respo... more Despite the relevance of adaptive immunity against equine pathogens antigen-specific T cell responses of horses are not well characterized and the lack of insight into T cell responses hampers the understanding of the pathogeneses of important diseases. In this study we used tetanus toxoid (TT) as a well-defined antigen to characterize antigen-reactive T cells. Six healthy adult horses received a routine booster against tetanus with an immune stimulating complex (ISCOM)-based vaccine and were followed for 28 days. TT-specific serum antibodies were quantified by ELISA and increased in all horses by day 7 after vaccination. CD154 is an established indicator of antigen-reactive T helper cells in other species, but has not been characterized in horses. CD154 detection in equine PBMC by an anti-human CD154 antibody (clone 5C8) was confirmed by Western blots and then applied for flow cytometry. As a common indicator of equine T cell activation, cytokine induction was studied in parallel. ...

Research paper thumbnail of New mAbs facilitate quantification of secreted equine TNF-α and flow cytometric analysis in monocytes and T cells

Veterinary Immunology and Immunopathology, 2021

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, that is involved in acute inflammation... more Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, that is involved in acute inflammation and is employed as a biomarker of inflammatory diseases in several species for which reliable quantification is available. We aimed to develop suitable tools to quantify TNF-α in equine samples. We generated two new mAbs against equine TNF-α (clones 48 and 292), evaluated their specificity for this cytokine, and confirmed detection of native TNF-α in stimulated equine PBMC. The TNF-α mAbs were paired in a fluorescent bead-based assay for quantification of equine TNF-α. The TNF-α assay had a wide quantification range of 12 pg/mL - 38.4 ng/mL. In addition, TNF-α mAb 48 was used for a detailed analysis of TNF-α production in PBMC by intracellular staining and flow cytometry. TNF-α was expressed by CD14+ monocytes after LPS stimulation and by monocytes and lymphocytes after polyclonal stimulation with PMA and ionomycin in vitro. TNF-α expressing lymphocytes consisted mainly of CD4+ T cells. CD8+ T cells and other lymphocytes also expressed TNF-α. The new mAbs evaluated here for soluble and intracellular TNF-α will enable the detailed analysis of this important pro-inflammatory cytokine during equine immune responses and inflammatory diseases of the horse.

Research paper thumbnail of Additional file 1: of Deletion of the ORF2 gene of the neuropathogenic equine herpesvirus type 1 strain Ab4 reduces virulence while maintaining strong immunogenicity

Figure S1. Cytokines in serum after infection with EHV-1 strain Ab4 or its deletion mutant Ab4ΔOR... more Figure S1. Cytokines in serum after infection with EHV-1 strain Ab4 or its deletion mutant Ab4ΔORF2. Horses (n = 8/group) were infected with one of two EHV-1 strains (Ab4) or Ab4ΔORF2 at day 0 or kept as uninfected controls. Serum was sampled at several times before (days − 8, − 1) and after (day 1- day 24) infection, cytokines and the inflammatory marker sCD14 were evaluated with fluorescent bead-based assays. Mean and standard errors for IFN-α (A), sCD14 (B), and IFN-γ (C) in the serum are displayed. Significant differences between groups are marked: a = Ab4 vs. control, b = Ab4ΔORF2 vs. control, c = Ab4 vs. Ab4ΔORF2. (JPG 332 kb)

Research paper thumbnail of Identification of Disease-Associated Cryptococcal Proteins Reactive With Serum IgG From Cryptococcal Meningitis Patients

Frontiers in Immunology, 2021

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment... more Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera fromC. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung funga...

Research paper thumbnail of RESEARCH ARTICLE Open Access Immune

response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent

Research paper thumbnail of Peripheral blood basophils are the main source for early interleukin-4 secretion upon in vitro stimulation with Culicoides allergen in allergic horses

PLOS ONE, 2021

Interleukin-4 (IL-4) is a key cytokine secreted by type 2 T helper (Th2) cells that orchestrates ... more Interleukin-4 (IL-4) is a key cytokine secreted by type 2 T helper (Th2) cells that orchestrates immune responses during allergic reactions. Human and mouse studies additionally suggest that basophils have a unique role in the regulation of allergic diseases by providing initial IL-4 to drive T cell development towards the Th2 phenotype. EquineCulicoideshypersensitivity (CH) is a seasonal immunoglobulin E (IgE)-mediated allergic dermatitis in horses in response to salivary allergens fromCulicoides(Cul) midges. Here, we analyzed IL-4 production in peripheral blood mononuclear cells (PBMC) of CH affected (n = 8) and healthy horses (n = 8) living together in an environment with naturalCulexposure. DuringCulexposure when allergic horses had clinical allergy, IL-4 secretion from PBMC after stimulation withCulextract was similar between healthy and CH affected horses. In contrast, allergic horses had higher IL-4 secretion from PBMC than healthy horses during months without allergen exposu...

Research paper thumbnail of An Equine Herpesvirus Type 1 (EHV-1) Ab4 Open Reading Frame 2 Deletion Mutant Provides Immunity and Protection from EHV-1 Infection and Disease

Journal of Virology, 2019

Nasal equine herpesvirus type 1 (EHV-1) shedding is essential for virus transmission during outbr... more Nasal equine herpesvirus type 1 (EHV-1) shedding is essential for virus transmission during outbreaks. Cell-associated viremia is a prerequisite for the most severe disease outcomes, abortion and equine herpesvirus myeloencephalopathy (EHM). Thus, protection from viremia is considered essential for preventing EHM. Ab4ΔORF2 vaccination prevented EHV-1 challenge virus replication in the upper respiratory tract in fully protected horses. Consequently, these neither shed virus nor developed cell-associated viremia. Protection from virus shedding and viremia during challenge infection in combination with reduced virulence at the time of vaccination emphasizes ORF2 deletion as a promising modification for generating an improved EHV-1 vaccine. During this challenge infection, full protection was linked to preexisting local and systemic EHV-1-specific antibodies combined with rapidly increasing intranasal IgG4/7 antibodies and lack of nasal type I interferon and chemokine induction. These h...

Research paper thumbnail of Deletion of the ORF2 gene of the neuropathogenic equine herpesvirus type 1 strain Ab4 reduces virulence while maintaining strong immunogenicity

BMC Veterinary Research, 2018

Background: Equine herpesvirus type 1 (EHV-1) induces respiratory infection, abortion, and neurol... more Background: Equine herpesvirus type 1 (EHV-1) induces respiratory infection, abortion, and neurologic disease with significant impact. Virulence factors contributing to infection and immune evasion are of particular interest. A potential virulence factor of the neuropathogenic EHV-1 strain Ab4 is ORF2. This study on 24 Icelandic horses, 2 to 4 years of age, describes the infection with EHV-1 Ab4, or its deletion mutant devoid of ORF2 (Ab4ΔORF2) compared to non-infected controls (each group n = 8). The horses' clinical presentation, virus shedding, viremia, antibody and cellular immune responses were monitored over 260 days after experimental infection. Results: Infection with Ab4ΔORF2 reduced fever and minimized nasal virus shedding after infection compared to the parent virus strain Ab4, while Ab4ΔORF2 established viremia similar to Ab4. Concurrently with virus shedding, intranasal cytokine and interferon α (IFN-α) production increased in the Ab4 group, while horses infected with Ab4ΔORF2 expressed less IFN-α. The antibody response to EHV-1 was evaluated by a bead-based multiplex assay and was similar in both infected groups, Ab4 and Ab4ΔORF2. EHV-1 specific immunoglobulin (Ig) G1 was induced 8 days after infection (d8 pi) with a peak on d10-12 pi. EHV-1 specific IgG4/7 increased starting on d10 pi, and remained elevated in serum until the end of the study. The intranasal antibody response to EHV-1 was dominated by the same IgG isotypes and remained elevated in both infected groups until d130 pi. In contrast to the distinct antibody response, no induction of EHV-1 specific T-cells was detectable by flow cytometry after ex vivo re-stimulation of peripheral blood mononuclear cells (PBMC) with EHV-1 in any group. The cellular immune response was characterized by increased secretion of IFN-γ and interleukin10 in response to ex vivo re-stimulation of PBMC with EHV-1. This response was present during the time of viremia (d5-10 pi) and was similar in both infected groups, Ab4 and Ab4ΔORF2. Conclusions: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa. In contrast, ORF2 does not influence viremia. The immunogenicity of the Ab4ΔORF2 and parent Ab4 viruses are identical.

Research paper thumbnail of C-C motif chemokine ligand (CCL) production in equine peripheral blood mononuclear cells identified by newly generated monoclonal antibodies

Veterinary Immunology and Immunopathology, 2018

Chemokines are soluble molecules directing immune cell trafficking and homing, mediating inflamma... more Chemokines are soluble molecules directing immune cell trafficking and homing, mediating inflammation, and initiating immune responses to infection. In horses, the analysis of chemokines has been limited by the lack of specific antibodies. We generated mAbs specific for the equine CC motif chemokine ligands (CCL) CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES) and CCL11 (eotaxin) using hybridoma technology. Antibody specificity was confirmed by intracellular staining of Chinese Hamster Ovary cells transfected with expression vectors encoding for CCL2, CCL3, CCL5, or CCL11. Transfectants were stained with the anti-CCL mAbs. Flow cytometric analysis confirmed the specificity of the different mAbs for the respective chemokine. In addition, equine PBMC were stained after isolation, culture in medium, or stimulation with LPS, or PMA and ionomycin. CCL2 was detected in few cluster of differentiation (CD)14 + monocytes in PBMC stimulated with PMA and ionomycin for 2 h. CCL3 was produced by CD14 + monocytes after 4-6 h culture in medium. After stimulation with PMA and ionomycin for 12-24 h, CCL3 was also expressed in lymphocytes, mainly in CD4 + T cells. Stimulation with LPS reduced the percentage of CCL3 + monocytes in PBMC. CCL5 was detected in PBMC ex vivo in CD4 + and CD8 + T cells. Culture of PBMC for longer than 6 h or stimulation with PMA and ionomycin reduced the percentage of CCL5 + cells. CCL11 was produced by CD4 + T cells in PBMC after stimulation with PMA and ionomycin for 4-24 h. After LPS stimulation of PBMC, CCL2, CCL5, and CCL11 production were comparable to culture in medium alone. ELISAs for each of the four chemokines were developed using pairs of anti-equine CCL mAbs. Supernatants from PMA and ionomycin stimulated PBMC contained detectable amounts of CCL2, CCL3 and CCL5, while CCL11 secretion could be stimulated from equine tracheal epithelial cells in response to IL-4. The newly generated mAbs for equine CCL chemokines facilitate the quantitative analysis of intracellular chemokine production by flow cytometry and soluble chemokines by ELISA. The CCL mAbs are valuable tools to improve the evaluation of innate immune responses in horses.

Research paper thumbnail of CXCL10 production in equine monocytes is stimulated by interferon-gamma

Veterinary Immunology and Immunopathology, 2018

C-X-C motif ligand 10 (CXCL10) is a pro-inflammatory chemokine and has been extensively evaluated... more C-X-C motif ligand 10 (CXCL10) is a pro-inflammatory chemokine and has been extensively evaluated in people and mice. In horses, CXCL10 and its involvement in host immunity has rarely been analyzed due to the lack of specific antibodies. We generated a mAb specific for the equine chemokine CXCL10 using hybridoma technology. Antibody specificity was confirmed by intracellular staining and flow cytometric analysis of Chinese Hamster Ovary (CHO) cells expressing equine rCXCL10, while CHO cells expressing equine rCXCL9 were not detected. Native CXCL10 expression in PBMC from horses of different age groups was analyzed by flow cytometry after in vitro stimulation. CXCL10 expressing PBMC were characterized by triple staining of CXCL10 combined with cell-surface markers. Stimulation with IFN-γ for 5 h similarly induced CXCL10 production in cluster of differentiation (CD) 14 + CD16 − MHCII high monocytes of adult horses and weanlings. The newly generated mAb enables the quantitative intracellular analysis of CXCL10 by flow cytometry and provides a new valuable tool to improve the evaluation of inflammatory responses in horses.

Research paper thumbnail of The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses

PLOS ONE, 2018

The equine herpesvirus type 1 (EHV-1) ORF1 and ORF71 genes have immune modulatory effects in vitr... more The equine herpesvirus type 1 (EHV-1) ORF1 and ORF71 genes have immune modulatory effects in vitro. Experimental infection of horses using virus mutants with multiple deletions including ORF1 and ORF71 showed promise as vaccine candidates against EHV-1. Here, the combined effects of ORF1 and ORF71 deletions from the neuropathogenic EHV-1 strain Ab4 on clinical disease and host immune response were further explored. Three groups of EHV-1 naïve horses were experimentally infected with the ORF1/71 gene deletion mutant (Ab4ΔORF1/71), the parent Ab4 strain, or remained uninfected. In comparison to Ab4, horses infected with Ab4ΔORF1/71 did not show the initial high fever peak characteristic of EHV-1 infection. Ab4ΔORF1/71 infection had reduced nasal shedding (1/5 vs. 5/5) and, simultaneously, decreased intranasal interferon (IFN)-α, interleukin (IL)-10 and soluble CD14 secretion. However, Ab4 and Ab4ΔORF1/71 infection resulted in comparable viremia, suggesting these genes do not regulate the infection of the mononuclear cells and subsequent viremia. Intranasal and serum anti-EHV-1 antibodies to Ab4ΔORF1/71 developed slightly slower than those to Ab4. However, beyond day 12 post infection (d12pi) serum antibodies in both virusinfected groups were similar and remained increased until the end of the study (d114pi). EHV-1 immunoglobulin (Ig) G isotype responses were dominated by short-lasting IgG1 and long-lasting IgG4/7 antibodies. The IgG4/7 response closely resembled the total EHV-1 specific antibody response. Ex vivo re-stimulation of PBMC with Ab4 resulted in IFN-γ and IL-10 secretion by cells from both infected groups within two weeks pi. Flow cytometric analysis showed that IFN-γ producing EHV-1-specific T-cells were mainly CD8 + /IFN-γ + and detectable from d32pi on. Peripheral blood IFN-γ + T-cell percentages were similar in both infected groups, albeit at low frequency (~0.1%). In summary, the Ab4ΔORF1/71 gene deletion mutant is less virulent but induced antibody responses and cellular immunity similar to the parent Ab4 strain.

Research paper thumbnail of Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay

Veterinary Immunology and Immunopathology, 2017

Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (I... more Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse. Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observed. The new IgA mAb 84-1 was used in combination with the previously characterized anti-equine IgA mAb BVS2 for the development and validation of a fluorescent bead-based assay to quantify total IgA in equine serum and various secretions. The IgA assay's linear detection ranged from 64 pg/ml to 1000 ng/ml. For the quantification of IgA in serum or in secretions an IgA standard was purified from serum or nasal wash fluid (secretory IgA), respectively. The different standards were needed for accurate IgA quantification in the respective samples taking the different signal intensities of monomeric and dimeric IgA on the florescent bead-based assay into account. IgA was quantified by the bead-based assay established here in different equine samples of healthy adult individuals. In serum the median total IgA was 0.45 mg/ml for Thoroughbred horses (TB, n = 10) and 1.16 mg/ ml in Icelandic horses (ICH, n = 12). In nasopharyngeal secretions of TB (n = 7) 0.13 mg/ml median total IgA was measured, and 0.25 mg/ml for ICH (n = 12). Saliva of ICH (n = 6) contained a median of 0.15 mg/ml, colostrum of Warmbloods (n = 8) a median of 1.89 mg/ml IgA. Compared to IgG1 and IgG4/7 quantified in the same samples, IgA appeared as the major immunoglobulin isotype in nasopharyngeal secretions and saliva while it is a minor isotype in serum and colostrum. The newly developed monoclonal antibodies against equine IgA and the resulting bead-based assay for quantification of total IgA can notably improve the evaluation of mucosal immunity in horses.

Research paper thumbnail of Influences of age and sex on leukocytes of healthy horses and their ex vivo cytokine release

Veterinary immunology and immunopathology, Jan 6, 2015

Leukocytes and their functional capacities are used extensively as biomarkers in immunological re... more Leukocytes and their functional capacities are used extensively as biomarkers in immunological research. Commonly employed indicators concerning leukocytes are as follows: number, composition in blood, response to discrete stimuli, cytokine release, and morphometric characteristics. In order to employ leukocytes as biomarkers for disease and therapeutic monitoring, physiological variations and influencing factors on the parameters measured have to be considered. The aim of this report was to describe the ranges of selected leukocyte parameters in a sample of healthy horses and to analyse whether age, sex, breed, and sampling time point (time of day) influence peripheral blood leukocyte composition, cell morphology and release of cytokines ex vivo. Flow cytometric comparative characterisation of cell size and complexity in 24 healthy horses revealed significant variance. Similarly, basal release of selected cytokines by blood mononuclear cells also showed high variability [TNFα (65-1...

Research paper thumbnail of Evaluation of the reactivity of commercially available monoclonal antibodies with equine cytokines

Veterinary Immunology and Immunopathology, 2013

Research paper thumbnail of Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent

BMC veterinary research, Jan 23, 2015

Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. ... more Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown. In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte num...