Allergenic Activity of Individual Cat Allergen Molecules.

More than 10% of the world's population suffers from an immunoglobulin E (IgE)-mediated allergy to cats which is accompanied mainly by respiratory symptoms such as rhinitis and asthma. Several cat allergen molecules have been identified, but their allergenic activity has not been investigated in depth. Purified cat allergen molecules (Fel d 1, Fel d 2, Fel d 3, Fel d 4, Fel d 6, Fel d 7 and Fel d 8) were characterized via mass spectrometry and circular dichroism spectroscopy regarding their molecular mass and fold, respectively. Cat-allergen-specific IgE levels were quantified via ImmunoCAP measurements in IgE-sensitized subjects with (n = 37) and without (n = 20) respiratory symptoms related to cat exposure. The allergenic activity of the cat allergens was investigated by loading patients' IgE onto rat basophils expressing the human FcεRI receptor and studying the ability of different allergen concentrations to induce ß-hexosaminidase release. Purified and folded cat allergens with correct masses were obtained. Cat-allergen-specific IgE levels were much higher in patients with a respiratory allergy than in patients without a respiratory allergy. Fel d 1, Fel d 2, Fel d 4 and Fel d 7 bound the highest levels of specific IgE and already-induced basophil degranulation at hundred-fold-lower concentrations than the other allergens. Fel d 1, Fel d 4 and Fel d 7 were recognized by more than 65% of patients with a respiratory allergy, whereas Fel d 2 was recognized by only 30%. Therefore, in addition to the major cat allergen Fel d 1, Fel d 4 and Fel d 7 should also be considered to be important allergens for the diagnosis and specific immunotherapy of cat allergy.

The impact of hybrid immunity on immune responses after SARS-CoV-2 vaccination in persons with multiple sclerosis treated with disease-modifying therapies.

BACKGROUND AND PURPOSE: Hybrid immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develops from a combination of natural infection and vaccine-generated immunity. Multiple sclerosis (MS) disease-modifying therapies (DMTs) have the potential to impact humoral and cellular immunity induced by SARS-CoV-2 vaccination and infection. The aims were to compare antibody and T-cell responses after SARS-CoV-2 mRNA vaccination in persons with MS (pwMS) treated with different DMTs and to assess differences between naïvely vaccinated pwMS and pwMS with hybrid immunity vaccinated following a previous SARS-CoV-2 infection. METHODS: Antibody and T-cell responses were determined in pwMS at baseline and 4 and 12 weeks after the second dose of SARS-CoV-2 vaccination in 143 pwMS with or without previous SARS-CoV-2 infection and 40 healthy controls (HCs). The MS cohort comprised natalizumab (n = 22), dimethylfumarate (n = 23), fingolimod (n = 38), cladribine (n = 30), alemtuzumab (n = 17) and teriflunomide (n = 13) treated pwMS. Immunoglobulin G antibody responses to SARS-CoV-2 antigens were measured using a multiplex bead assay and FluoroSpot was used to assess T-cell responses (interferon γ and interleukin 13). RESULTS: Humoral and T-cell responses to vaccination were comparable between naïvely vaccinated HCs and pwMS treated with natalizumab, dimethylfumarate, cladribine, alemtuzumab and teriflunomide, but were suppressed in fingolimod-treated pwMS. Both fingolimod-treated pwMS and HCs vaccinated following a previous SARS-CoV-2 infection had higher antibody levels 4 weeks after vaccination compared to naïvely vaccinated individuals. Antibody and interferon γ levels 12 weeks after vaccination were positively correlated with time from last treatment course of cladribine. CONCLUSION: These findings are of relevance for infection risk mitigation and for vaccination strategies amongst pwMS undergoing DMT.

Cross-reactive EBNA1 immunity targets alpha-crystallin B and is associated with multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system, for which and Epstein-Barr virus (EBV) infection is a likely prerequisite. Due to the homology between Epstein-Barr nuclear antigen 1 (EBNA1) and alpha-crystallin B (CRYAB), we examined antibody reactivity to EBNA1 and CRYAB peptide libraries in 713 persons with MS (pwMS) and 722 matched controls (Con). Antibody response to CRYAB amino acids 7 to 16 was associated with MS (OR = 2.0), and combination of high EBNA1 responses with CRYAB positivity markedly increased disease risk (OR = 9.0). Blocking experiments revealed antibody cross-reactivity between the homologous EBNA1 and CRYAB epitopes. Evidence for T cell cross-reactivity was obtained in mice between EBNA1 and CRYAB, and increased CRYAB and EBNA1 CD4+ T cell responses were detected in natalizumab-treated pwMS. This study provides evidence for antibody cross-reactivity between EBNA1 and CRYAB and points to a similar cross-reactivity in T cells, further demonstrating the role of EBV adaptive immune responses in MS development.

Measurement of Horse Allergens Equ c 1 and Equ c 2: A Comparison among Breeds.

INTRODUCTION: Horse allergens are less studied than allergens from other furry animals and these allergens must be evaluated to understand the complexity of allergy to horses. The aims of this study were to develop assays for the horse allergens Equ c 1 and Equ c 2 in dander and saliva and to determine their levels in ten horse breeds. The study also included a comparison of these findings with previous results on the levels of Equ c 4 performed on the same study population. METHOD: The study population included 170 horses from 10 horse breeds including American Curly and Russian Bashkir horse, which have been suggested to be hypoallergenic. Competitive ELISA assays were developed, with polyclonal antibodies as capture antibodies, for the detection of Equ c 1 and Equ c 2 in dander and saliva samples. RESULTS: The horse allergens Equ c 1 and Equ c 2 were found in all dander and saliva samples from the ten horse breeds. The GM level (ng/µg protein) of Equ c 1 in dander was 470 (range 129-2,569) and in saliva samples, 40 (range 6-160). The GM level of Equ c 2 in dander was 138 (range 18-1,650) and in saliva samples, 0.8 (range 0.03-17). In dander, there were no significant differences in Equ c 1 and Equ c 2 GM levels between stallions, mares, and geldings. CONCLUSION: Our results show high intra- and inter-breed variability. Neither the American Curly horse nor the Russian Bashkir horse, earlier categorized as hypoallergenic breeds, was associated with lower allergen levels of Equ c 1, Equ c 2, or Equ c 4 than the other horse breeds investigated.

Molecular Allergen-Specific IgE Recognition Profiles and Cumulative Specific IgE Levels Associated with Phenotypes of Cat Allergy.

Cat allergy is a major trigger factor for respiratory reactions (asthma and rhinitis) in patients with immunoglobulin E (IgE) sensitization. In this study, we used a comprehensive panel of purified cat allergen molecules (rFel d 1, nFel d 2, rFel d 3, rFel d 4, rFel d 7, and rFel d 8) that were obtained by recombinant expression in Escherichia coli or by purification as natural proteins to study possible associations with different phenotypes of cat allergy (i.e., rhinitis, conjunctivitis, asthma, and dermatitis) by analyzing molecular IgE recognition profiles in a representative cohort of clinically well-characterized adult cat allergic subjects (n = 84). IgE levels specific to each of the allergen molecules and to natural cat allergen extract were quantified by ImmunoCAP measurements. Cumulative IgE levels specific to the cat allergen molecules correlated significantly with IgE levels specific to the cat allergen extract, indicating that the panel of allergen molecules resembled IgE epitopes of the natural allergen source. rFel d 1 represented the major cat allergen, which was recognized by 97.2% of cat allergic patients; however, rFel d 3, rFel d 4, and rFel d 7 each showed IgE reactivity in more than 50% of cat allergic patients, indicating the importance of additional allergens in cat allergy. Patients with cat-related skin symptoms showed a trend toward higher IgE levels and/or frequencies of sensitization to each of the tested allergen molecules compared with patients suffering only from rhinitis or asthma, while there were no such differences between patients with rhinitis and asthma. The IgE levels specific to allergen molecules, the IgE levels specific to cat allergen extract, and the IgE levels specific to rFel d 1 were significantly higher in patients with four different symptoms compared with patients with 1-2 symptoms. This difference was more pronounced for the sum of IgE levels specific to the allergen molecules and to cat extract than for IgE levels specific for rFel d 1 alone. Our study indicates that, in addition to rFel d 1, rFel d 3, rFel d 4, and rFel d 7 must be considered as important cat allergens. Furthermore, the cumulative sum of IgE levels specific to cat allergen molecules seems to be a biomarker for identifying patients with complex phenotypes of cat allergy. These findings are important for the diagnosis of IgE sensitization to cats and for the design of allergen-specific immunotherapies for the treatment and prevention of cat allergy.

Recombinant multimeric dog allergen prevents airway hyperresponsiveness in a model of asthma marked by vigorous TH 2 and TH 17 cell responses.

BACKGROUND: Allergy to dogs affects around 10% of the population in developed countries. Immune therapy of allergic patients with dog allergen extracts has shown limited therapeutic benefit. METHODS: We established a mouse model of dog allergy by repeatedly administering dog dander and epithelium extracts via the intranasal route. We also assessed the efficacy of a recombinant multimeric protein containing Can f 1, f 2, f 4 and f 6 in preventing inflammatory responses to dog extracts. RESULTS: Repeated inhalation of dog extracts induced infiltration of the airways by TH 2 cells, eosinophils and goblet cells, reminiscent of the house dust mite (HDM) model of asthma. Dog extracts also induced robust airway hyperresponsiveness and promoted TH 17 cell responses, which was associated with a high neutrophilic infiltration of the airways. scRNA-Seq analysis of T helper cells in the airways pinpointed a unique gene signature for TH 17 cells. Analysis of T-cell receptors depicted a high frequency of clones that were shared between TH 17, TH 2 and suppressive Treg cells, indicative of a common differentiation trajectory for these subsets. Importantly, sublingual administration of multimeric Can f 1-2-4-6 protein prior to sensitization reduced airway hyperresponsiveness and type 2-mediated inflammation in this model. CONCLUSION: Dog allergen extracts induce robust TH 2 and TH 17 cell-mediated responses in mice. Recombinant Can f 1-2-4-6 can induce tolerance to complex dog allergen extracts.

Allergic sensitization to lipocalins reflects asthma morbidity in dog dander sensitized children.

Background: Sensitization to dog is an important risk factor for asthma in children, but the clinical relevance of IgE to available dog- and furry animal allergen molecules is uncertain. Methods: Spirometry, methacholine challenge, fraction of exhaled nitric oxide, nasal challenge with dog extract and questionnaires were performed in 59 dog-sensitized children (age 10-18 years). Serum IgE to dog-, cat-, horse extracts and the allergen molecules Can f 1-6, Fel d 1, Fel d 2, Fel d 4 and Equ c 1 were evaluated. Results: Median numbers of positive IgE results to furry animal allergen molecules among children without asthma was 3, with asthma 5.5 and with troublesome asthma 9 (asthma vs. no asthma; p = 0.039; troublesome asthma vs. no asthma; p = 0.009). The odds ratio for asthma if sensitized to any lipocalin was 7.2 (95% confidence Interval: 1.44-35.9). Children with troublesome asthma had higher IgE levels to the lipocalins Can f 2, Can f 4 and Can f 6 compared to the rest of the study population (44 vs. 4.1 kUA/L, p = 0.015; 5.8 vs. 0.9 kUA/L, p = 0.018 and 1.3 vs. 0.7 kUA/L, p = 0.03 respectively). Furthermore, a positive nasal challenge was more common among children with troublesome asthma (83% vs. 36%, p = 0.036). Conclusions: Polysensitization to furry animal allergens and lipocalins is associated with asthma in dog-sensitized children. Children with troublesome asthma have higher IgE levels to several dog lipocalins than other dog sensitized children. Key message: Polysensitization to furry animal allergens and high IgE levels to the dog lipocalins Can f 2, Can f 4 and Can f 6 is associated with asthma severity in dog dander sensitized children. Molecular allergy diagnostics may thus help the clinicians to evaluate the impact of allergic sensitization on asthma morbidity.

Generation of Tumor-Specific Cytotoxic T Cells From Blood via In Vitro Expansion Using Autologous Dendritic Cells Pulsed With Neoantigen-Coupled Microbeads.

For the past decade, adoptive cell therapy including tumor-infiltrating lymphocytes, genetically modified cytotoxic lymphocytes expressing a chimeric antigen receptor, or a novel T-cell receptor has revolutionized the treatment of many cancers. Progress within exome sequencing and neoantigen prediction technologies provides opportunities for further development of personalized immunotherapies. In this study, we present a novel strategy to deliver in silico predicted neoantigens to autologous dendritic cells (DCs) using paramagnetic beads (EpiTCer beads). DCs pulsed with EpiTCer beads are superior in enriching for healthy donor and patient blood-derived tumor-specific CD8+ T cells compared to DC loaded with whole-tumor lysate or 9mer neoantigen peptides. A dose-dependent effect was observed, with higher EpiTCer bead per DC being favorable. We concluded that CD8+ T cells enriched by DC loaded with EpiTCer beads are tumor specific with limited tumor cross-reactivity and low recognition of autologous non-activated monocytes or CD8+ T cells. Furthermore, tumor specificity and recognition were improved and preserved after additional expansion using our Good Manufacturing Process (GMP)-compatible rapid expansion protocol. Phenotypic analysis of patient-derived EpiTCer DC expanded CD8+ T cells revealed efficient maturation, with high frequencies of central memory and effector memory T cells, similar to those observed in autologous expanded tumor-infiltrating lymphocytes. These results indicate that DC pulsed with EpiTCer beads enrich for a T-cell population with high capacity of tumor recognition and elimination, which are features needed for a T-cell product to be used for personalized adoptive cell therapy.

Identification of four novel T cell autoantigens and personal autoreactive profiles in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), in which pathological T cells, likely autoimmune, play a key role. Despite its central importance, the autoantigen repertoire remains largely uncharacterized. Using a novel in vitro antigen delivery method combined with the Human Protein Atlas library, we screened for T cell autoreactivity against 63 CNS-expressed proteins. We identified four previously unreported autoantigens in MS: fatty acid-binding protein 7, prokineticin-2, reticulon-3, and synaptosomal-associated protein 91, which were verified to induce interferon-γ responses in MS in two cohorts. Autoreactive profiles were heterogeneous, and reactivity to several autoantigens was MS-selective. Autoreactive T cells were predominantly CD4+ and human leukocyte antigen-DR restricted. Mouse immunization induced antigen-specific responses and CNS leukocyte infiltration. This represents one of the largest systematic efforts to date in the search for MS autoantigens, demonstrates the heterogeneity of autoreactive profiles, and highlights promising targets for future diagnostic tools and immunomodulatory therapies in MS.

Development of humoral and cellular immunological memory against SARS-CoV-2 despite B cell depleting treatment in multiple sclerosis.

B cell depleting therapies (BCDTs) are widely used as immunomodulating agents for autoimmune diseases such as multiple sclerosis. Their possible impact on development of immunity to severe acute respiratory syndrome virus-2 (SARS-CoV-2) has raised concerns with the coronavirus disease 2019 (COVID-19) pandemic. We here evaluated the frequency of COVID-19-like symptoms and determined immunological responses in participants of an observational trial comprising several multiple sclerosis disease modulatory drugs (COMBAT-MS; NCT03193866) and in eleven patients after vaccination, with a focus on BCDT. Almost all seropositive and 17.9% of seronegative patients on BCDT, enriched for a history of COVID-19-like symptoms, developed anti-SARS-CoV-2 T cell memory, and T cells displayed functional similarity to controls producing IFN-γ and TNF. Following vaccination, vaccine-specific humoral memory was impaired, while all patients developed a specific T cell response. These results indicate that BCDTs do not abrogate SARS-CoV-2 cellular memory and provide a possible explanation as to why the majority of patients on BCDTs recover from COVID-19.

Oligodendrocyte myelin glycoprotein as a novel target for pathogenic autoimmunity in the CNS.

Autoimmune disorders of the central nervous system (CNS) comprise a broad spectrum of clinical entities. The stratification of patients based on the recognized autoantigen is of great importance for therapy optimization and for concepts of pathogenicity, but for most of these patients, the actual target of their autoimmune response is unknown. Here we investigated oligodendrocyte myelin glycoprotein (OMGP) as autoimmune target, because OMGP is expressed specifically in the CNS and there on oligodendrocytes and neurons. Using a stringent cell-based assay, we detected autoantibodies to OMGP in serum of 8/352 patients with multiple sclerosis, 1/28 children with acute disseminated encephalomyelitis and unexpectedly, also in one patient with psychosis, but in none of 114 healthy controls. Since OMGP is GPI-anchored, we validated its recognition also in GPI-anchored form. The autoantibodies to OMGP were largely IgG1 with a contribution of IgG4, indicating cognate T cell help. We found high levels of soluble OMGP in human spinal fluid, presumably due to shedding of the GPI-linked OMGP. Analyzing the pathogenic relevance of autoimmunity to OMGP in an animal model, we found that OMGP-specific T cells induce a novel type of experimental autoimmune encephalomyelitis dominated by meningitis above the cortical convexities. This unusual localization may be directed by intrathecal uptake and presentation of OMGP by meningeal phagocytes. Together, OMGP-directed autoimmunity provides a new element of heterogeneity, helping to improve the stratification of patients for diagnostic and therapeutic purposes.

The allergenic activity and clinical impact of individual IgE-antibody binding molecules from indoor allergen sources.

A large number of allergens have been discovered but we know little about their potential to induce inflammation (allergenic activity) and symptoms. Nowadays, the clinical importance of allergens is determined by the frequency and intensity of their IgE antibody binding (allergenicity). This is a rather limited parameter considering the development of experimental allergology in the last 20 years and the criteria that support personalized medicine. Now it is known that some allergens, in addition to their IgE antibody binding properties, can induce inflammation through non IgE mediated pathways, which can increase their allergenic activity. There are several ways to evaluate the allergenic activity, among them the provocation tests, the demonstration of non-IgE mediated pathways of inflammation, case control studies of IgE-binding frequencies, and animal models of respiratory allergy. In this review we have explored the current status of basic and clinical research on allergenic activity of indoor allergens and confirm that, for most of them, this important property has not been investigated. However, during recent years important advances have been made in the field, and we conclude that for at least the following, allergenic activity has been demonstrated: Der p 1, Der p 2, Der p 5 and Blo t 5 from HDMs; Per a 10 from P. americana; Asp f 1, Asp f 2, Asp f 3, Asp f 4 and Asp f 6 from A. fumigatus; Mala s 8 and Mala s 13 from M. sympodialis; Alt a 1 from A. alternata; Pen c 13 from P. chrysogenum; Fel d 1 from cats; Can f 1, Can f 2, Can f 3, Can f 4 and Can f 5 from dogs; Mus m 1 from mice and Bos d 2 from cows. Defining the allergenic activity of other indoor IgE antibody binding molecules is necessary for a precision-medicine-oriented management of allergic diseases.

Sensitive detection of antigen-specific T-cells using bead-bound antigen for in vitro re-stimulation.

Reliable and sensitive detection of antigen specific cells is essential in several fields of research, whether it concerns monitoring responses to infectious agents or exploring the auto-antigen repertoire in autoimmune diseases. Identification of these cells is however difficult, especially when the cells often are rare and methods not sensitive, specific or practical enough. We propose a novel method of processing antigens before stimulation of cells which consists of covalently binding protein antigen to superparamagnetic micro-beads and using denaturing washes to remove contaminants. Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated using both cytomegalovirus and tetanus-diphtheria antigen-beads as well as non-antigenic protein-beads as negative control in an IFNγ FluoroSpot assay in order to detect Th1 and CD8+ responses. The responses toward the antigen beads were both antigen specific and sensitive, with a detection threshold of 1 IFNγ producing T-cell per 18,000 PBMCs. •Covalently binding antigen to paramagnetic beads allows for harsh denaturing washes without loss of antigen.•Microbeads are phagocytosed by antigen presenting cells, resulting in efficient uptake, processing and presentation of the antigens.•The method allows the usage of relatively impure starting antigen material and whole PBMC samples without high background levels in follow up cellular assays.

A hypoallergenic peptide mix containing T cell epitopes of the clinically relevant house dust mite allergens.

BACKGROUND: In the house dust mite (HDM) Dermatophagoides pteronyssinus, Der p 1, 2, 5, 7, 21, and 23 have been identified as the most important allergens. The aim of this study was to define hypoallergenic peptides derived from the sequences of the six allergens and to use the peptides and the complete allergens to study antibody, T cell, and cytokine responses in sensitized and nonsensitized subjects. METHODS: IgE reactivity of HDM-allergic and non-HDM-sensitized individuals to 15 HDM allergens was established using ImmunoCAP ISAC technology. Thirty-three peptides covering the sequences of the six HDM allergens were synthesized. Allergens and peptides were tested for IgE and IgG reactivity by ELISA and ImmunoCAP, respectively. Allergenic activity was determined by basophil activation. CD4+ T cell and cytokine responses were determined in PBMC cultures by CFSE dilution and Luminex technology, respectively. RESULTS: House dust mite allergics showed IgE reactivity only to complete allergens, whereas 31 of the 33 peptides lacked relevant IgE reactivity and allergenic activity. IgG antibodies of HDM-allergic and nonsensitized subjects were directed against peptide epitopes and higher allergen-specific IgG levels were found in HDM allergics. PBMC from HDM-allergics produced higher levels of IL-5 whereas non-HDM-sensitized individuals mounted higher levels of IFN-gamma, IL-17, pro-inflammatory cytokines, and IL-10. CONCLUSION: IgG antibodies in HDM-allergic patients recognize peptide epitopes which are different from the epitopes recognized by IgE. This may explain why naturally occurring allergen-specific IgG antibodies do not protect against IgE-mediated allergic inflammation. A mix of hypoallergenic peptides containing T cell epitopes of the most important HDM allergens was identified.

Plasma protein profiling reveals candidate biomarkers for multiple sclerosis treatment.

Multiple sclerosis (MS) treatment options have improved significantly over the past decades, but the consequences of MS can still be devastating and the needs for monitoring treatment surveillance are considerable. In the current study we used affinity proteomics technology to identify potential biomarkers which could ultimately be used to as facilitate treatment decisions. We profiled the intra-individual changes in the levels of 59 target proteins using an antibody suspension bead array in serial plasma samples from 44 MS patients during treatment with natalizumab followed by fingolimod. Nine proteins showed decreasing plasma levels during natalizumab treatment, with PEBP1 and RTN3 displaying the most significant changes. Protein levels remained stable during fingolimod treatment for both proteins. The decreasing PEBP1 levels during natalizumab treatment could be validated using ELISA and replicated in an independent cohort. These results support the use of this technology as a high throughput method of identifying potentially useful biomarkers of MS treatment.

Myelin oligodendrocyte glycoprotein revisited-sensitive detection of MOG-specific T-cells in multiple sclerosis.

Autoreactive CD4+ T-cells are believed to be a main driver of multiple sclerosis (MS). Myelin oligodendrocyte glycoprotein (MOG) is considered an autoantigen, yet doubted in recent years. The reason is in part due to low frequency and titers of MOG autoantibodies and the challenge to detect MOG-specific T-cells. In this study we aimed to analyze T-cell reactivity and frequency utilizing a novel method for detection of antigen-specific T-cells with bead-bound MOG as stimulant. Peripheral blood mononuclear cells (PBMCs) from natalizumab treated persons with MS (n = 52) and healthy controls (HCs) (n = 24) were analyzed by IFNγ/IL-22/IL-17A FluoroSpot. A higher number of IFNγ (P = 0.001), IL-22 (P = 0.003), IL-17A (P < 0.0001) as well as double and triple cytokine producing MOG-specific T-cells were detected in persons with MS compared to HCs. Of the patients, 46.2-59.6% displayed MOG-reactivity. Depletion of CD4+ T-cells or monocytes or blocking HLA-DR completely eliminated the MOG specific response. Anti-MOG antibodies did not correlate with T-cell MOG-responses. In conclusion, we present a sensitive method to detect circulating autoreactive CD4+ T-cells producing IFNγ, IL-22 or IL-17A using MOG as a model antigen. Further, we demonstrate that MOG-specific T-cells are present in approximately half of persons with MS.