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.
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.
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-α molecule containing a foreign immunogenic T helper epitope, and the induction of neutralizing anti-TNF-α autoantibodies was analysed. These mice were then sensitized with ovalbumin (OVA), and the effect of neutralizing anti-TNF-α autoantibodies on the allergen-induced airway inflammation was analysed. Results: AutoVac TNF106-immunized mice developed high titres of neutralizing anti-TNF-α 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-α 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-α autoantibody production by the AutoVac TNF106 technology not only confirmed the role of TNF-α 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.
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-alpha, 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-alpha 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.
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.
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