BACKGROUND:Cytokines play an integral role in the coordination and persistence of allergic inflammatory processes and therefore represent prime targets for novel therapies in diseases such as asthma. Multiple attempts to generate low-molecular-weight cytokine inhibitors have failed, and the main attention has focused on the development of biological agents such as neutralizing antibodies. The present work describes a simple and effective method to induce the production of therapeutic anti-cytokine autoantibodies by active immunization against a modified endogenous cytokine. METHODS:Balb/c mice were subcutaneously injected with AutoVac TNF106, a recombinant murine TNF-alpha molecule containing a foreign immunogenic T helper epitope, and the induction of neutralizing anti-TNF-alpha autoantibodies was analysed. These mice were then sensitized with ovalbumin (OVA), and the effect of neutralizing anti-TNF-alpha autoantibodies on the allergen-induced airway inflammation was analysed. RESULTS:AutoVac TNF106-immunized mice developed high titres of neutralizing anti-TNF-alpha autoantibodies, which were maintained for at least 4 weeks after the last booster injection. Mice vaccinated with AutoVac TNF106 and further immunized against OVA showed diminished TNF-alpha levels in the bronchoalveolar lavage (BAL) fluid after OVA challenge. Moreover, pretreatment with AutoVac TNF106 resulted in significantly reduced numbers of eosinophils and neutrophils in BAL fluid in response to single or multiple allergen exposure. CONCLUSION:The induction of anti-TNF-alpha autoantibody production by the AutoVac TNF106 technology not only confirmed the role of TNF-alpha in the induction of allergic inflammation but also offers a novel approach to block the activity of cytokines in order to treat allergic inflammatory conditions.

Current therapeutic approaches to asthma have had limited impact on the clinical management and resolution of this disorder. By using a novel vaccine strategy targeting the inflammatory cytokine IL-5, we have ameliorated hallmark features of asthma in mouse models. Delivery of a DNA vaccine encoding murine IL-5 modified to contain a promiscuous foreign Th epitope bypasses B cell tolerance to IL-5 and induces neutralizing polyclonal anti-IL-5 Abs. Active vaccination against IL-5 reduces airways inflammation and prevents the development of eosinophilia, both hallmark features of asthma in animal models and humans. The reduced numbers of inflammatory T cells and eosinophils in the lung also result in a marked reduction of Th2 cytokine levels. Th-modified IL-5 DNA vaccination reduces the expression of IL-5 and IL-4 by approximately 50% in the airways of allergen-challenged mice. Most importantly, Th-modified IL-5 DNA vaccination restores normal bronchial hyperresponsiveness to beta-methacholine. Active vaccination against IL-5 reduces key pathological events associated with asthma, such as Th2 cytokine production, airways inflammation, and hyperresponsiveness, and thus represents a novel therapeutic approach for the treatment of asthma and other allergic conditions.

Current therapeutic approaches to asthma have had limited impact on the clinical management and resolution of this disorder. By using a novel vaccine strategy targeting the inflammatory cytokine IL-5, we have ameliorated hallmark features of asthma in mouse models. Delivery of a DNA vaccine encoding murine IL-5 modified to contain a promiscuous foreign Th epitope bypasses B cell tolerance to IL-5 and induces neutralizing polyclonal anti-IL-5 Abs. Active vaccination against IL-5 reduces airways inflammation and prevents the development of eosinophilia, both hallmark features of asthma in animal models and humans. The reduced numbers of inflammatory T cells and eosinophils in the lung also result in a marked reduction of Th2 cytokine levels. Th-modified IL-5 DNA vaccination reduces the expression of IL-5 and IL-4 by ∼50% in the airways of allergen-challenged mice. Most importantly, Th-modified IL-5 DNA vaccination restores normal bronchial hyperresponsiveness to β-methacholine. Active vaccination against IL-5 reduces key pathological events associated with asthma, such as Th2 cytokine production, airways inflammation, and hyperresponsiveness, and thus represents a novel therapeutic approach for the treatment of asthma and other allergic conditions.

We have used retroviral vector technology to develop a method for functional screening of combinatorial peptide libraries expressed inside mammalian cells with the ultimate goal of identifying new drug targets. The method was validated in a library screening experiment based on antigen presentation of small peptides. A library encoding SIXNXEKX-peptides, where X designates randomised positions corresponding to major histocompatibility (MHC) class I anchor residues, was generated in a retroviral vector. The library was transduced into a population of antigen presenting cells (APCs) known to mediate MHC class I restricted presentation of the SIINFEKL peptide. The cellular library was screened by using an antigen presentation assay in which a T cell hybridoma recognising the MHC class I/SIINFEKL peptide complex was employed. Using this experimental model, we identified two positive cellular clones both encoding SIINFEKL peptides with identical codon usage. This number corresponded well to the expected frequency of SIINFEKL in the library. The lack of identification of other peptides capable of activating the T-hybridoma supports previous findings of a high degree of specificity at the level of peptide-loading of MHC-molecules. The result further demonstrates the potential of using combinatorial libraries for functional screening and selection of effector peptides stably expressed in mammalian cells.

A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B‐cell epitopes in two different proteins using a peptide‐based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15‐mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor‐α, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non‐covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor‐α were added to identify potential linear B‐cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non‐covalent binding although the peptides were in fact present on the non‐covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B‐cell epitopes in protein antigens. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.

BioTechniquesVol. 26, No. 6 BenchmarksOpen AccessFunctional Testing of a Bicistronic Retroviral Vector for Intracellular Peptide ProductionM. Duch, A. Tolstrup, I. Dalum, T. Jespersen, S. Mouritsen & F.S. PedersenM. DuchUniversity of Aarhus, Aarhus, Denmark, A. TolstrupUniversity of Aarhus, Aarhus, Denmark, I. DalumUniversity of Aarhus, Aarhus, Denmark, T. JespersenUniversity of Aarhus, Aarhus, Denmark, S. MouritsenUniversity of Aarhus, Aarhus, Denmark & F.S. PedersenUniversity of Aarhus, Aarhus, DenmarkPublished Online:22 Aug 2018https://doi.org/10.2144/99266bm04AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByBreaking B-cell Tolerance and CTL Tolerance in three OVA-transgenic Mouse Strains Expressing Different Levels of OVAScandinavian Journal of Immunology, Vol. 67, No. 2Generation of Autoreactive CTL by Tumour Vaccines Containing Foreign T Helper EpitopesScandinavian Journal of Immunology, Vol. 65, No. 3Linked Foreign T-Cell Help Activates Self-Reactive CTL and Inhibits Tumor Growth21 June 2005 | The Journal of Immunology, Vol. 175, No. 1Activation of p53 by scaffold-stabilised expression of Mdm2-binding peptides: visualisation of reporter gene induction at the single-cell level21 September 2004 | British Journal of Cancer, Vol. 91, No. 8Tapasin enhances MHC class I peptide presentation according to peptide half-life30 July 2004 | Proceedings of the National Academy of Sciences, Vol. 101, No. 32Functional screening of a retroviral peptide library for MHC class I presentationGene, Vol. 263, No. 1-2 Vol. 26, No. 6 Follow us on social media for the latest updates Metrics Downloaded 43 times History Published online 22 August 2018 Published in print June 1999 Information© 2018 Author(s)PDF download

Tumor necrosis factor-alpha (TNF-alpha) is critically involved in the pathogenesis of several chronic inflammatory diseases. Monoclonal antibodies against TNF-alpha are currently used for the treatment of rheumatoid arthritis and Crohn's disease. This report describes a simple and effective method for active immunization against self TNF-alpha. This vaccination approach leads to a T-cell-dependent polyclonal and sustainable anti-TNF-alpha autoantibody response that declines upon discontinuation of booster injections. The autoantibodies are elicited by injecting modified recombinant TNF-alpha molecules containing foreign immunodominant T-helper epitopes. In mice immunized with such molecules, the symptoms of experimental cachexia and type II collagen-induced arthritis are ameliorated. These results suggest that vaccination against TNF-alpha may be a useful approach for the treatment of rheumatoid arthritis and other chronic inflammatory diseases.

Self proteins are handled in the same way as foreign proteins by antigen presenting cells, but because of T-cell tolerance the presentation of self peptides does not normally lead to T cell activation. By providing physically linked T-cell help it is possible to overcome the B cell non-responsiveness toward self antigens. We have shown previously that a very potent antibody response, cross-reactive with a self protein, can be rapidly induced by immunizing with a recombinant immunogen consisting of the self protein with a foreign immunodominant T helper epitope inserted into its sequence (Dalum, I., Jensen, M. R., Hindersson, P., Elsner, H. I. and Mouritsen, S. (1996) J. Immnunol. 157, 4796). In this study we compare this approach for inducing autoantibodies against a self protein with the traditional method of conjugating the self antigen to a foreign carrier protein. The highly conserved self protein ubiquitin with an inserted epitope from ovalbumin (UbiOVA) is used as a model protein and compared to two traditionally conjugated immunogens consisting of ubiquitin chemically conjugated to a peptidic T helper epitope or to ovalbumin. The traditionally conjugated immunogens induce much slower and low titered ubiquitin specific antibody responses than the recombinant construct which also is capable of inducing antibodies directed against a much broader range of potential ubiquitin B cell determinants than the chemically conjugated immunogens. All three constructs are processed by antigen presenting cells and ovalbumin derived T cell epitopes are presented to T helper cells. From these observations it seems likely that the presence of non-shielded autologous B cell determinants on the immunogen is critical for the ability to induce a strong autoantibody response with a diverse fine specificity. Furthermore, the ubiquitin specific antibodies induced by UbiOVA contain higher levels of IgG2a/b relative to IgG1 compared to the conjugates. We therefore speculate that the insertion of a T cell epitope directly into the self antigen could possibly induce an immune response with a different Th1/Th2 balance than a response induced with traditional conjugates.

Self proteins are processed and presented by APCs in the same way as foreign proteins. Presentation of fragments derived from self proteins does not, however, lead to Th cell stimulation because of T cell tolerance. In this study, a novel approach was used to investigate whether B cell tolerance toward a self Ag could be due to the absence of this Th cell recognition. The highly conserved nonimmunogenic protein ubiquitin was used as a model protein. Two modified ubiquitin molecules were constructed with ubiquitin segments exchanged either with the T cell epitope, OVA(325-336), which binds to the mouse A(d) MHC class II molecule, or with the T cell epitope, hen egg lysozyme(50-61), which binds to the A(k) molecule. Mice were immunized with the resulting proteins. Both modified proteins elicited strong autoantibody responses toward soluble native ubiquitin, demonstrating that insertion of a single foreign T cell epitope can overcome the B cell nonresponsiveness. The T cell regulatory role of one of the inserted foreign T cell epitopes in ubiquitin was studied, and at least two different Th cell specificities were found to operate in the response. The T cells were directed against: 1) the inserted epitope, and 2) a combination of the inserted epitope and parts of the neighboring ubiquitin regions. Therefore, the absence of T cell help seems to be an important reason for B cell tolerance toward self proteins.

More than 90% of the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APC) have in their binding site a peptide derived from an extracellular protein ingested by the APC or from a protein of the APC itself. These self-peptides can be eluted from affinity-purified MHC class II molecules by acid elution, and have been studied with a variety of techniques. We show here that the self-peptides eluted from the mouse MHC class II molecules Ad, Ed and Ek bind specifically to MHC class II molecules of the allelic type from which they were derived. The pH optimum for binding is around 5.0, i.e. the same optimum at which synthetic peptides representing sequences of foreign antigens bind to MHC class II molecules. This suggests that the physiological compartment where MHC class II molecules bind self-peptides may be very late in the endocytic pathway. The chemical properties of the eluted and labelled MHC class II peptides were studied by isoelectric focusing. This method was able to separate the peptides very efficiently, and enabled a rapid comparison of peptides eluted from different MHC molecules. The 125I-labelled peptides displayed a broad range of isoelectric points with values predominantly below neutral. This suggests that such peptides bind to MHC in a predominantly non-charged state.