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.

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.

Cotranslational folding cooperativity of contiguous domains of α-spectrin.

Proteins synthesized in the cell can begin to fold during translation before the entire polypeptide has been produced, which may be particularly relevant to the folding of multidomain proteins. Here, we study the cotranslational folding of adjacent domains from the cytoskeletal protein α-spectrin using force profile analysis (FPA). Specifically, we investigate how the cotranslational folding behavior of the R15 and R16 domains are affected by their neighboring R14 and R16, and R15 and R17 domains, respectively. Our results show that the domains impact each other's folding in distinct ways that may be important for the efficient assembly of α-spectrin, and may reduce its dependence on chaperones. Furthermore, we directly relate the experimentally observed yield of full-length protein in the FPA assay to the force exerted by the folding protein in piconewtons. By combining pulse-chase experiments to measure the rate at which the arrested protein is converted into full-length protein with a Bell model of force-induced rupture, we estimate that the R16 domain exerts a maximal force on the nascent chain of ∼15 pN during cotranslational folding.

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.

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.

Allergens in dog extracts: Implication for diagnosis and treatment.

BACKGROUND: Five to ten percent of the population in affluent countries are allergic to dog. Diagnosis and treatment is based on allergen extracts from natural sources where composition and concentration are poorly defined. OBJECTIVE: We aimed to quantify six dog allergens (Can f 1-6) in commercial skin prick test (SPT) solutions and to determine individual allergen profiles in dogs. METHOD: The allergen content of SPT solutions from five vendors and allergen source material from three anatomical sites were analyzed. Fur and saliva samples were collected from a mixed population of 120 dogs. Can f 1-6 were quantified by inhibition ELISA using purified recombinant or natural allergens and polyclonal or monoclonal antibodies. Allergenicity was analyzed by basophil activation test. RESULTS: Extensive variation in allergen composition was observed in commercial SPT vials resulting in a patient-dependent ability to activate basophils. Extract heterogeneity depended on collection site and allergen composition in individual dogs and source materials. Can f 2 and Can f 6 exhibited low levels in fur and SPT solutions, whereas Can f 4, which was the dominating allergen in fur samples, did not display similar high proportions in SPT solutions. Can f 3 varied most among SPT solutions. CONCLUSION: There is a great variation of dog allergens in natural extracts raising questions of source, sampling, processing and ultimately of standardization and minimum allergen levels for accurate diagnosis and treatment.

Cotranslational folding of spectrin domains via partially structured states.

How do the key features of protein folding, elucidated from studies on native, isolated proteins, manifest in cotranslational folding on the ribosome? Using a well-characterized family of homologous α-helical proteins with a range of biophysical properties, we show that spectrin domains can fold vectorially on the ribosome and may do so via a pathway different from that of the isolated domain. We use cryo-EM to reveal a folded or partially folded structure, formed in the vestibule of the ribosome. Our results reveal that it is not possible to predict which domains will fold within the ribosome on the basis of the folding behavior of isolated domains; instead, we propose that a complex balance of the rate of folding, the rate of translation and the lifetime of folded or partly folded states will determine whether folding occurs cotranslationally on actively translating ribosomes.

Trigger Factor Reduces the Force Exerted on the Nascent Chain by a Cotranslationally Folding Protein.

Cotranslational protein folding can generate pulling forces on the nascent chain that can affect the instantaneous translation rate and thereby possibly feed back on the folding process. Such feedback would represent a new way of coupling translation and folding, different from coupling based on, for example, codon usage. However, to date, we have carried out the experiments used to measure pulling forces generated by cotranslational protein folding either in reconstituted in vitro translation systems lacking chaperones, in ill-defined cell lysates, or in vivo; hence, the effects of chaperones on force generation by folding are unknown. Here, we have studied the cotranslational folding of dihydrofolate reductase (DHFR) in the absence and in the presence of the chaperones trigger factor (TF) and GroEL/ES. DHFR was tethered to the ribosome via a C-terminal linker of varying length, ending with the SecM translational arrest peptide that serves as an intrinsic force sensor reporting on the force generated on the nascent chain when DHFR folds. We find that DHFR folds into its native structure only when it has emerged fully outside the ribosome and that TF and GroEL alone substantially reduces the force generated on the nascent chain by the folding of DHFR, while GroEL/ES has no effect. TF therefore weakens the possible coupling between cotranslational folding and translation.

Cotranslational Protein Folding inside the Ribosome Exit Tunnel.

At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins.

Designing a multimer allergen for diagnosis and immunotherapy of dog allergic patients.

BACKGROUND: Dog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality. OBJECTIVE: To assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog. METHODS: A linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production. RESULTS: The linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses. CONCLUSION: We provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.

Mammalian-derived respiratory allergens - implications for diagnosis and therapy of individuals allergic to furry animals.

Furry animals cause respiratory allergies in a significant proportion of the population. A majority of all mammalian allergens are spread as airborne particles, and several have been detected in environments where furry animals are not normally kept. The repertoire of allergens from each source belongs to a restricted number of allergen families. Classification of allergen families is particularly important for the characterization of allergenicity and cross-reactivity of allergens. In fact, major mammalian allergens are taken from only three protein families, i.e. the secretoglobin, lipocalin and kallikrein families. In particular, the lipocalin superfamily harbours major allergens in all important mammalian allergen sources, and cross-reactivity between lipocalin allergens may explain cross-species sensitization between mammals. The identification of single allergen components is of importance to improve diagnosis and therapy of allergic patients using component-resolved diagnostics and allergen-specific immunotherapy (ASIT) respectively. Major disadvantages with crude allergen extracts for these applications emphasize the benefits of careful characterization of individual allergens. Furthermore, detailed knowledge of the characteristics of an allergen is crucial to formulate attenuated allergy vaccines, e.g. hypoallergens. The diverse repertoires of individual allergens from different mammalian species influence the diagnostic potential and clinical efficacy of ASIT to furry animals. As such, detailed knowledge of individual allergens is essential for adequate clinical evaluation. This review compiles current knowledge of the allergen families of mammalian species, and discusses how this information may be used for improved diagnosis and therapy of individuals allergic to mammals.

High basophil allergen sensitivity (CD-sens) is associated with severe allergic asthma in children.

Children with problematic severe asthma (PA) have persistent symptoms and/or severe exacerbations despite treatment with several drugs. Classification of asthma severity is currently based on level of treatment and assessment of asthma control, but objective biomarkers of asthma severity are needed. To investigate the clinical relevance of basophil allergen threshold sensitivity (CD-sens) as a measure of allergen sensitivity in a well-characterized cohort of children with different manifestations of persistent allergic asthma. Cat-allergic children (6-18 yr) with problematic severe asthma (n = 11) according to GINA were compared with eleven age-matched children with controlled, but persistent asthma (CA). The protocol included standardized questionnaires, asthma control test (ACT), spirometry, methacholine challenges, measurement of FE(NO,) IgE, cat IgE and IgG antibodies, and analysis of CD-sens (CD63-expression) by flow cytometry. The 11 cat-allergic children with PA had a significantly lower ACT score (p < 0.001), reduced FEV(1) (p = 0.04), and increased numbers of blood eosinophils (p = 0.03) compared with the 11 children with CA. The former exhibited a higher CD-sens to cat (p = 0.02). No significant differences were detected with respect to FE(NO) (p = 0.17), IgE (p = 0.84), cat IgE (p = 0.12), and the major cat-allergen rFel d 1 (p = 0.30). CD-sens significantly correlated with ACT (p = 0.002, r = -0.63) and FE(NO) (p = 0.01, r = 0.55). No significant differences between PA and CA were found regarding IgG antibodies to rFel d 1. Cat-allergic children with problematic severe asthma have higher sensitivity to cat allergen, as measured by CD-sens, compared with children with controlled asthma. This suggests that CD-sens could be used as an additional marker for identifying children with the most severe allergic asthma.

Covalent coupling of vitamin D3 to the major cat allergen Fel d 1 improves the effects of allergen-specific immunotherapy in a mouse model for cat allergy.

BACKGROUND: Allergen-specific immunotherapy (SIT) is currently the only curative treatment for allergy but the treatment needs to be improved. We hypothesize that covalent coupling of immunomodulating vitamin D3 to the major cat allergen Fel d 1 can enhance the beneficial effects of SIT to cat allergy. METHODS: We treated mice sensitized to Fel d 1 with subcutaneous injections of two doses of recombinant Fel d 1 coupled to 1α,25-dihydroxyvitamin D3 (rFel d 1:VD3) and compared to treatment with the same doses of rFel d 1 in a mouse model for cat allergy. Airway hyperresponsiveness (AHR), cytokines and cells in bronchoalveolar lavage (BAL), in vitro activation of splenocytes to rFel d 1, and Fel d 1-specific immunoglobulins were evaluated. RESULTS: Treatment with both doses of rFel d 1:VD3 decreased AHR, cellular influx and Th2 cytokines in BAL compared to untreated mice. High- and low-dose rFel d 1 treatment also decreased AHR and BAL Th2 cytokines, with less decrease for the low-dose treatment. Importantly, the total number of cells and eosinophils in BAL was markedly reduced at both high- and low-dose rFel d 1:VD3 compared to treatment with rFel d 1 alone. Finally, treatment with both rFel d 1 and rFel d 1:VD3 induced Fel d 1-specific serum IgG. CONCLUSION: Our results indicate a beneficial therapeutic effect of rFel d 1:VD3 on airway inflammation, AHR and rFel d 1-specific immune responses and thus suggest that this novel immunomodulatory candidate may improve both the efficacy and safety of SIT.

In vitro evolution of allergy vaccine candidates, with maintained structure, but reduced B cell and T cell activation capacity.

Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients.

Crystal structure of the dog lipocalin allergen Can f 2: implications for cross-reactivity to the cat allergen Fel d 4.

The dog lipocalin allergen Can f 2 is an important cause of allergic sensitization in humans worldwide. Here, the first crystal structure of recombinant rCan f 2 at 1.45 A resolution displays a classical lipocalin fold with a conserved Gly-Xaa-Trp motif, in which Trp19 stabilizes the overall topology of the monomeric rCan f 2. Phe38 and Tyr84 localized on the L1 and L5 loops, respectively, control access to the highly hydrophobic calyx. Although the rCan f 2 calyx is nearly identical with the aero-allergens MUP1, Equ c 1 and A2U from mouse, horse and rat, respectively, no IgE cross-reactivity was found using sera from five mono-sensitized subjects. However, clear IgE cross-reactivity was demonstrated between Can f 2 and the cat allergen Fel d 4, although they share less than 22% sequence identity. This suggests a role for these allergens in co-sensitization between cat- and dog-allergic patients.

Production, crystallization and preliminary X-ray diffraction analysis of the allergen Can f 2 from Canis familiaris.

The allergen Can f 2 from dog (Canis familiaris) present in saliva, dander and fur is an important cause of allergic sensitization worldwide. Here, the production, isolation, crystallization and preliminary X-ray diffraction analysis of two crystal forms of recombinant Can f 2 are reported. The first crystal form belonged to space group C222, with unit-cell parameters a = 68.7, b = 77.3, c = 65.1 A, and diffracted to 1.55 A resolution, while the second crystal form belonged to space group C2, with unit-cell parameters a = 75.7, b = 48.3, c = 68.7 A, beta = 126.5 degrees , and diffracted to 2.1 A resolution. Preliminary data analysis indicated the presence of a single molecule in the asymmetric unit for both crystal forms.