Anand Gautam

Active Vaccination Against IL-5 Bypasses Immunological Tolerance and Ameliorates Experimental Asthma

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.

HER-2 DNA and Protein Vaccines Containing Potent Th Cell Epitopes Induce Distinct Protective and Therapeutic Antitumor Responses in HER-2 Transgenic Mice

Overexpression of the growth factor receptor HER-2 (c-erbB-2, neu) has transforming potential and occurs in ∼20–30% of breast and ovarian cancers. HER-2 is a self Ag, but Abs and T cells specific for HER-2 have been isolated from cancer patients, suggesting HER-2 may be a good target for active immunotherapy. We constructed rat HER-2 DNA and protein vaccines containing potent Th cell epitopes derived from tetanus toxin and studied their potency in two strains of mice transgenic for the rat HER-2 molecule. Vaccination with HER-2 DNA protected nontransgenic mice from tumor challenge, but induced only moderate protection in one of the tumor models. However, vaccination with the modified HER-2 protein resulted in almost complete protection from tumor challenge in both tumor models. This protection could be mediated by Abs alone. In addition, protein vaccination efficiently eliminated pre-established tumors in both models, even when vaccination occurred 9 days after tumor implantation. These data demonstrate the potential of HER-2-based vaccines as therapeutic agents for the treatment of cancers overexpressing HER-2.

Induction of TNF-α Autoantibody Production by AutoVac TNF106: A Novel Therapeutic Approach for the Treatment of Allergic Diseases

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.

Linked Foreign T-Cell Help Activates Self-Reactive CTL and Inhibits Tumor Growth

Transgenic mice expressing membrane-bound OVA under the rat insulin promoter, RIP-mOVA, has previously been suggested to display deletional tolerance toward the dominant CTL epitope, SIINFEKL, and provide an elegant model system to test the hypothesis that the lack of T cell help contributes to the tolerance. To understand how the CD8 tolerance is maintained in these mice, a set of neo-self-Ags, OVA, modified to contain a foreign Th peptide, were constructed and tested for their ability to induce CTL responses in RIP-mOVA mice. Immunization with these Th peptide-modified OVA molecules and not with the wild-type OVA induced self-reactive CTLs recognizing dominant CTL peptide, SIINFEKL. Importantly, immunization with the modified OVA constructs also prevented the growth of OVA-expressing tumors in transgenic mice. Since endogenous OVA Th peptides did not contribute toward breaking self CTL tolerance, these results also highlighted a very robust CD4 T cell tolerance toward OVA in RIP-mOVA mice that has not been previously described. These results therefore provide direct evidence that it is the tolerance in the CD4 Th cell compartment that helps maintain the CTL tolerance against self-Ag in these mice. Since the CTL tolerance can be broken or bypassed by foreign Th peptides inserted into a self Ag, potential of using this approach in generating effective therapeutic cancer vaccines is discussed.

Generation of Autoreactive CTL by Tumour Vaccines Containing Foreign T Helper Epitopes

The aim of this study was to evaluate the effect of including a foreign T helper cell epitope in vaccines designed for generation of CTL against self‐antigens and for inhibition of tumour growth. Two different vaccine designs were composed, a minimal epitope vaccine and a modified full length self‐antigen, both based on OVA containing either a colinearily synthesized or an inserted Th‐epitope, respectively. These vaccines were used for immunization of tolerant OVA transgenic mice (RIP‐OVAlow) and non‐tolerant C57BL/6 mice. First, it was shown that transgenic mice were tolerant to OVA in the CD4 compartment. Secondly, only the vaccines containing the foreign Th‐epitope and not the wild‐type constructs were able to induce self‐reactive CTL in the transgenic mice. Thirdly, these self‐reactive CTL induced by the Th‐epitope modified constructs also inhibited tumour growth in the OVA transgenic mice. Overall, these results demonstrate that inclusion of a foreign Th‐epitope circumvents the tolerance in this OVA transgenic strain. In addition, these results show the importance of including strong T‐cell help in cancer vaccines.

Breaking B‐cell Tolerance and CTL Tolerance in three OVA‐transgenic Mouse Strains Expressing Different Levels of OVA

It is of major importance to overcome the immunological tolerance in attempts to generate efficient tumour vaccines. Here, we describe induction of autoantibodies and self‐reactive CTL in three types of OVA‐transgenic mouse strains, RIP‐OVAlow, RIP‐mOVA and RIP‐OVAHI exhibiting varying levels of OVA expression and tolerance. This was achieved by immunizing with DNA constructs where a foreign T‐helper epitope, P30 from tetanus toxin, was inserted into the OVA sequence. OVA wild‐type DNA as well as the P30‐modified OVA DNA vaccines (OVA‐P30) were constructed and used for immunization in the OVA‐transgenic mouse strains as well as in control C57Bl/6 mice. The data show that insertion of a foreign T‐helper peptide (P30) in OVA is sufficient for breaking B‐cell tolerance in three different OVA‐transgenic mice strain. This approach is sufficient for induction of self‐reactive CTL in two of the three strains that expressed either a membrane‐bound form of OVA or a low amount of soluble OVA. It was not possible to induce CTL but still possible to induce autoantibodies in the strain that expressed a higher level of soluble OVA.

Nonobese Diabetic Mice Display Elevated Levels of Class II-Associated Invariant Chain Peptide Associated with I-A g7 on the Cell Surface

Peptide presentation by MHC class II molecules plays a pivotal role in determining the peripheral T cell repertoire as a result of both positive and negative selection in the thymus. Homozygous I-Ag7 expression imparts susceptibility to autoimmune diabetes in the nonobese diabetic mouse, and recently, it has been proposed that this arises from ineffectual peptide binding. Following biosynthesis, class II molecules are complexed with class II-associated invariant chain peptides (CLIP), which remain associated until displaced by Ag-derived peptides. If I-Ag7 is a poor peptide binder, then this may result in continued occupation by CLIP to the point of translocation to the cell surface. To test this hypothesis we generated affinity-purified polyclonal antisera that recognized murine CLIP bound to class II molecules in an allele-independent fashion. We have found abnormally high natural levels of cell surface class II occupancy by CLIP on nonobese diabetic splenic B cells. Experiments using I-A-transfected M12.C3 cells showed that I-Ag7 alone was associated with elevated levels of CLIP, suggesting that this was determined solely by the amino acid sequence of the class II molecule. These results indicated that an intrinsic property of I-Ag7 would affect both the quantity and the repertoire of self-peptides presented during thymic selection.

EAE: A Model for Immune Intervention with Synthetic Peptides

The cellular and molecular requirements for the autoimmune disease EAE are being defined in increasing detail through intense scrutiny of critical autoantigenic peptides, class II MHC molecules, and alpha beta TCRs involved in the disease process. This study has led to novel immunotherapeutic approaches, many of which are based on the administration of synthetic peptides. Since short peptides are understood to be the minimal antigenic units bound by MHC molecules for recognition by T cells, they are attractive experimental tools for finely modulating specific immune responses. It is clear that a large number of defined peptides can dramatically influence the course of EAE. Table IV lists a number of potential mechanisms which may mediate disease prevention. Increasing evidence supports the idea that prevention of autoimmune disease can result from MHC-blockade by peptides which competitively bind to class II molecules. However, for some peptides such as the perplexing partial agonist Ac1-11[4A], the mechanism by which these precisely defined units act is not yet fully understood. Numerous hurdles hinder immediate clinical application of peptide-based immunotherapy. Nevertheless, the knowledge gained by probing experimental autoimmunity with defined peptides promises to inspire original and practical approaches to treating human autoimmune disease.