Description of the Biomodulation Therapy of autoimmune diseases
Peptide-based therapeutic vaccines for allergic and autoimmune diseases
Today’s western world has rating number of patiens with allergic and autoimmune diseases which are forms of immune hypersensitivity that increasingly cause chronic ill health.
Most current therapies treat symptoms rather than addressing underlying immunological mechanisms. The ability to modify antigen-specific pathogenic responses by therapeutic vaccination offers the prospect of targeted therapy resulting in long-term clinical improvement without nonspecific immune suppression as we see it on daily basis in everyday practice of routine allergologist or reumatologist.
Currently used immunosupression has vast consequence for the overall homeostastis of organism and all the medical world knows that it is not a solution, despite it applies.
Examples of specific immune modulation can be found in nature and in established forms of immune desensitization. Current understanding and exploiting common mechanisms such as the ability to induce antigen-specific regulatory cells should allowed the development of effective therapeutic strategies for both forms of immunopathology. Targeting pathogenic T cells using vaccines consisting of synthetic peptides representing T cell epitopes is one such strategy that is currently being used with encouraging results.
Future challenges in the development of therapeutic vaccines include selection of appropriate antigens and peptides, optimization of peptide dose and route of administration and identifying strategies to induce bystander suppression.
Allergic and autoimmune diseases are manifestations of immunological hypersensitivity. They arise when mechanisms controlling responses to innocuous environmental antigens (such as allergens), or to endogenous host ('self') proteins, break down.
Analysis of genes contributing to allergic and autoimmune disorders has shown that susceptibility arises from complex multigenic interactions. Many genetic elements controlling immune responses are polymorphic. Notably, genes encoding major histocompatibility complex (MHC; also known as human leukocyte antigens or HLA) class I and II are strongly associated with certain autoimmune diseases.
Such associations support a prominent role for T cells in the recognition of a restricted number of epitopes derived from autologous or cross-reactive exogenous proteins (such as viral proteins). Interestingly, despite the well-described role of T cells in the pathogenesis of allergic inflammation, few HLA-allergic disease associations exist. Explanations for the difference between allergy and autoimmunity may include the fact that responses to self-epitopes are tightly regulated through central and peripheral tolerance mechanisms.
In contrast, responses to exogenous proteins, containing many HLA-binding epitopes, are not regulated to the same extent, particularly through central tolerance.
The dramatic recent increase in the prevalence of allergic sensitization and autoimmune diseases, particularly in industrialized countries, provides evidence for the additional role of environmental factors in the pathogenesis of immune hypersensitivity.
Improved sanitation, mass vaccination programs and widespread use of antibiotics have been associated not only with reduced burden of infectious disease, but also with declining immunological interaction with microorganisms.
Reduced exposure to predominantly T helper type 1 (TH1) response−inducing environmental stimuli was initially proposed to explain the increased incidence of allergic sensitization ('hygiene hypothesis'). But parallel increases in the prevalence of TH1-mediated autoimmune diseases such as type 1 diabetes implies immunological dysfunction common to both TH1- and TH2-mediated diseases. Clinical studies have shown the coexistence of both TH1- and TH2-mediated mechanisms in asthma. Thus, it seems unlikely that deficiency in TH1 or TH2 responses drives pathology associated with the other. Nor is it likely that simple, mutual antagonism of TH1 and TH2 responses in autoimmunity and allergy represent viable therapeutic approaches. Indeed, experimental models have shown the potential for exacerbation of disease through this strategy.
The hygiene hypothesis, in its original form, did not incorporate mechanisms of immune regulation common to TH1 and TH2 processes.
Thus, alternative explanations for the increased occurrence of disease may include the simple concept of clonal competition, in which hypersensitivity may be passively controlled by homeostatic regulation arising through expansion of immune cells specific for prevalent infectious agents, a simple competition for scarce resources precluding hyper-reactivity to innocuous antigens10.
In addition, it seems likely that regulatory mechanisms are established during early life in response to childhood infections and exposure to environmental organisms and their products.
Data, derived predominantly from experimental animal models, show that distinct populations of immunoregulatory T cells limit, or protect organisms from, immune pathology. A number of subsets have been described, including 'natural' CD4+ CD25+ cells and those expressing the transcription factor FoxP3, an apparent marker of regulatory potential. TH3 cells synthesizing transforming growth factor (TGF)- and Tr1 cells secreting interleukin (IL)-10 have been described in human models. Functional deficits in CD25+ and Tr1 subsets of regulatory cells have been reported in both allergic and autoimmune diseases. Whether these observations reflect an intrinsic defect in the regulatory cells themselves (a concept difficult to reconcile with the antigen-selective nature of these diseases), or their inability to control overexuberant effector responses, remains to be established.
Current treatment: the unmet need
Current pharmacologic treatments for autoimmune and allergic disorders (such as glucocorticosteroids, cyclophosphamide, methotrexate and antihistamines) are largely palliative (rather than curative) and some may result in nonspecific immunosuppression.
This may be associated with a range of complications including infections, the development of tumors and disruption of natural regulatory mechanisms. Long-term use of palliative drugs is associated with significant compliance and economic issues.
For these reasons, there is renewed enthusiasm for strategies aiming to reinstate homeostasis toward environmental and self-antigens. Here we discuss how peptide-based 'therapeutic vaccines' may recover immunological tolerance through the induction of immunoregulatory T cells and explain how current vaccines for the state of the art custom made therapy are designed.
Background to the approach of therapeutic vaccination
There is evidence that both allergic and autoimmune pathology, resulting from deficient immune regulation, is reversible. Allergic children can 'grow out' of food allergies, autoimmune diseases such as rheumatoid arthritis can transiently resolve without intervention during pregnancy and 'specific' allergen immunotherapy (SIT) (or in some cases, natural exposure to allergen) can modulate the antigen-specific immune response, resulting in long-term disease modification. Identifying common elements of these processes provide the basis for therapeutic interventions for autoimmune diseases .
Treatment of allergic rhinitis and asthma with specific immunotherapy is of proven efficacy and has been practiced for almost a century. Subcutaneous or sublingual administration of the sensitizing protein(s) modifies immunity and reduces allergen sensitivity. Mechanistic studies show downregulation of TH2 responses in peripheral blood or increased TH1 responses in the tissue, or both.
Recently, increased numbers of cells containing mRNA encoding IL-10 (and in some cases TGF-) or the proteins themselves have been reported in blood and tissues of treated individuals. Moreover, IL-10−secreting regulatory T cells are known to suppress pathology in experimental models of autoimmune diseases. Further evidence that the resolution of allergic disease may involve factors other than simple translation of TH2 responses to TH1 has been obtained from several models. Beekeepers exposed to multiple stings are protected from severe allergic reactions through a mechanism involving IL-10−secreting T cells. IL-10 has immunosuppressive functions on both T cells and mast cells. As with SIT for venom and aeroallergen sensitivity, protection of beekeepers is associated with induction of IgG isotypes, particularly IgG4, an isotype selectively promoted by IL-10.
Cross-linking of allergen-specific IgE on the surface of mast cells and basophils leads to activation and degranulation, with the release of mediators such as histamine and leukotrienes. Furthermore, IgE on the surface of antigen-presenting cells (APC) and B cells enhances uptake of allergen (allergen focusing) for presentation to T cells. IgG may downregulate the allergic response by competition with IgE for allergen binding (classical 'blocking antibody' theory), prevention of aggregation of receptor-bound IgE through steric hindrance, or by interference with antigen focusing by IgE bound to APC. Interestingly, a major role for mast cells has recently been described in the pathogenesis of autoimmune diseases, providing further evidence to support common mechanisms in allergic and autoimmune pathology. Related mechanisms in pathogenesis are likely to be amenable to common approaches in therapy.
Historically, increased TH1:TH2 cytokine ratios and induction of IgG focused therapeutic strategies in allergy on exploitation of the mutual antagonism of TH1 and TH2 responses, a concept adopted in reverse, with respect to autoimmune disease.
For example, glatiramer acetate (also known as GA, Cop-1, Copaxone), one of the most widely prescribed drugs for multiple sclerosis, is a random amino acid copolymer (poly (YEAK)n) known to activate T cells specific for myelin basic protein (MBP) and induce TH2 responses.
These responses may relate to the occurrence of adverse events of an allergic nature, such as flushing, urticaria, pruritis and chest tightness.
Glatiramer acetate binds to several MHC molecules including HLA-DRB1*1501 (encoded by a disease-associated allele in multiple sclerosis) in vitro. It has been suggested that glatiramer acetate competes with MBP epitopes for presentation to T cells, reducing activation of MBP-specific clones.
Recently, modified copolymers based on similar sequences (VWAK and FYAK), blocked native epitope binding to MHC class II in a mouse model of multiple sclerosis, polarized T-cell cytokine responses toward a 'TH2' profile and generated regulatory populations of T cells secreting IL-4 and IL-10 that were able to transfer protection from disease.
Furthermore, defined synthetic 15-mers (peptides of 15 amino acids in length), have been designed based on the HLA-binding characteristics of glatiramer acetate and the immunodominant native epitope MBP83−99.
Peptides with high affinity for HLA-DRB1*1501 suppressed autoimmune encephalomyelitis .Protection was associated with production of IL-4 and IL-10 by splenocytes and lymph node cells.
In human studies and increasingly in experimental models, IL-10 is being regarded as an immunosuppressive or regulatory cytokine. Thus, autoimmune processes may be ameliorated by the induction of a combination of TH2 (IL-4) and regulatory responses (IL-10, TGF-), just as allergic disease can be addressed by the induction of both TH1 (interferon (IFN)-) and immunoregulation (IL-10, TGF-). conditions.
Mucosal administration of antigen frequently results in downregulation of the immune response and is associated with the induction of regulatory cells. In 1829, Dakin observed a means by which the cutaneous hypersensitivity to poisonous vegetables such as poison oak and poison ivy could be avoided; "mystical, marvellous physicians, or favoured ladies with knowledge inherent, say the bane will prove the best antidote, and hence advice the forbidden leaves to be eaten, both as preventive and cure to the external disease." This was one of the earliest recorded examples of mucosal tolerance, a phenomenon that waited more than 150 years for the discovery of its mechanism.
Mucosal tolerance induction has been evaluated in numerous experimental models of allergy and autoimmune disease, but clinical data from trials in humans have been generally disappointing. Trials of oral tolerance involved extracts of myelin in multiple sclerosis, retinal extract for autoimmune uveitis, bovine collagen in rheumatoid arthritis and insulin in type 1 diabetes.
Relatively low doses of antigen were administered but did not provide significant clinical benefit. In fact, administration of a complex mixture of antigens in uveitis was associated with worsening of disease.
Regulatory T cells have been shown to have an important role in mucosal tolerance, with effector phenotype depending upon the nature of the antigen. Thus, protein antigens tend to induce TGF-−secreting TH3 cells, whereas short peptide antigens elicit IL-10−secreting Tr1 cells. Importantly, regulatory cells induced through mucosal tolerance have been shown to mediate bystander (or 'intermolecular') suppression, a process through which regulatory cells specific for one protein suppress the response of nearby effector cells to another protein. The latter seems to require copresentation of the two antigens by the same APC.
Bystander suppression is an important feature of mucosal and other forms of antigen-induced suppression because, it may override the phenomenon of 'epitope spreading.'
Epitope spreading was first described as a complication of autoimmune disease whereby the initiating immune response expands with time to include responses to other antigens. The issue of epitope spreading has been raised as a potential barrier to the development of effective immunological strategies to combat autoimmune diseases.
Therapeutic vaccines must have the potential to modulate pathogenic responses to several antigens, as responses to individual proteins may wax and wane within an individual. Ideally, effective vaccines should generate a network of intramolecular ('linked') and intermolecular ('bystander') suppression, such that tolerance induced to one protein (MBP, for example) can be exploited to regulate responses to others (proteolipid protein, for example).
Nature of the antigen for an effective therapeutic vaccine
The aim of a therapeutic vaccine is to achieve effective modulation of immune responses. Administration of the intact antigen would avoid having to select specific epitopes to suit MHC-disparate individuals. But intact antigen can activate mast cells and basophils, by cross-linking IgE in allergic individuals, as well as activating pathogenic B and T cells in both allergic and autoimmune diseases.
Peptide-based therapeutic vaccines
As short linear peptide sequences generally lack the ability to cross link adjacent IgE molecules on mast cells and basophils, a particular advantage of synthetic T cell epitopes in allergic disease is the avoidance of IgE-mediated activation. Early promise with peptide therapy in experimental models of allergy led to evaluation of synthetic peptides for immunotherapy in clinical trials
In general, peptide therapy was shown to reduce sensitivity to allergen and downregulate allergen-specific proliferative and cytokine responses in the blood. In studies evaluating treatment with allergen peptides, reduced skin reactivity to allergen was accompanied by decreased TH1 and TH2 responses in the blood and increases in IL-10. Furthermore, airway hyperreactivity was reduced in asthmatic subjects and nasal symptom scores improved in subjects with allergic rhinitis.
Two recent studies evaluating peptides derived from heat-shock proteins for the treatment of diabetes and rheumatoid arthritis have provided encouraging results. Individuals with newly diagnosed type 1 diabetes were treated by subcutaneous injection at three time points
After 10 months, islet cell function had been maintained in the treated group but had declined in placebo controls. In the treated group, peripheral blood T cells produced more IL-10 and IL-13, indicating modulation toward a TH2-regulatory phenotype.
Altered peptide ligands - The use of altered peptide ligands (APL) or T cell antagonists as potential vaccines for autoimmune disease.
Such peptides share MHC binding characteristics with the native peptide sequence but, as a result of amino acid substitutions (particularly in residues that contact the T cell receptor), deliver antagonist or partial agonist signals, modifying T cell activation and cytokine production.
The dosage of antagonist peptide
In human studies, even ultra-low doses of peptide have been shown to modulate T cell responses. Intradermal injection of microgram quantities of synthetic allergen peptides induced transient activation of antigen-specific T cells in the airways. In asthmatic individuals, this was manifested as bronchoconstriction measurable a few hours after injection. In these individuals, transient T cell activation was followed by months of marked and enduring hyporesponsivness, which has recently been linked to the induction of regulatory CD4+ T cells. Similar transient activation before tolerance was described in a number of mouse experimental models. The occurrence of contrast-enhancing lesions during peptide therapy in multiple sclerosis may also be related to transient effector T cell activation. With allergen peptides, induction of bronchoconstriction was not required for the ensuing peptide-induced hyporesponsiveness. Although this does not preclude low-level activation of antigen-specific T cells before systemic tolerance induction, it implies that activation of effector T cells is not an absolute requirement for tolerance. Thus, the magnitude of the initial effector T cell response may be controlled (for example, by reducing dose) to avoid T cell−dependent disease exacerbations such as bronchoconstriction in asthma and brain lesions in multiple sclerosis.
Differences in protein or peptide dose may not translate into a substantially different pharmacologically active dose. The route of administration, physical and biological half-life, and solubility of the tolerogen probably have roles in determining the active dose (and the immunological context of that dose) reaching the blood and lymphatics. For example, microgram doses of allergen peptides administered intradermally resulted in systemic manifestations of tolerance, whereas the same preparation delivered by inhalation (nebulized in saline and inhaled orally) did not. This observation suggests that the route of administration can determine tolerogenicity and that transient activation of effector T cells may be dissociated from ensuing tolerance because it was possible to transiently activate effector T cells in the airways through both routes. It may be that a threshold plasma dose of tolerogen must be achieved to establish systemic tolerance, even in low-dose regimens. Although intradermal or intravenous administration may require the lowest doses to achieve this, the inconvenience of injection may favor higher-dose regimens using intranasal delivery, which has been shown to lead to rapid systemic delivery in experimental models
Appropriate selection of epitopes and corresponding peptides for therapeutic vaccines is crucial for success. Translation of findings in murine models is complicated by the use of inbred strains of mice. Polymorphism of the genes encoding human MHC class I and II presents a challenge for peptide vaccine design. In autoimmune diseases, HLA associations with disease provide a natural platform for vaccine design once target antigens have been identified.
Whereas few diseases (such as ankylosing spondylitis) are overwhelmingly linked to the genes encoding HLA, a small number of epitopes (capable of binding to the disease-associated HLA molecule) selected from the most important target proteins may form the basis of an effective peptide vaccine.
The lack of HLA association in allergic diseases, coupled in some cases with prolific polymorphism in target allergens, initially presents a more complex scenario. But the few detailed studies of the MHC-binding characteristics of primary sequence from both allergens and parasite antigens, indicates that relatively short regions of sequence contain multiple overlapping MHC-binding motifs. On this basis it has been possible to design peptide vaccines based on a small number of MHC-binding peptides that theoretically provide coverage of the majority of the population (assuming that a minimum of one epitope must be recognized to achieve a biological response)
The role of APCs
Further important features of an effective therapeutic peptide vaccine are the ability to bind to class II MHC proteins in a conformation that mimics the naturally processed epitope of the self-antigen or allergen, together with solubility of peptide components and the lack of an associated innate immune response. Peptides that are insoluble can induce local inflammation. Soluble peptides can bind directly to dendritic cells (DCs) in lymphoid tissues and may activate regulatory T cells. Alloreactive human T cells stimulated with mature DCs produce IL-2 and IFN- and proliferate strongly. In contrast, a number of studies in mice and humans have shown that antigens carried by immature DCs induce IL-10−secreting T cells. Repetitive in vitro stimulation of alloreactive human T cells with immature DCs generated anergic regulatory T cells secreting IL-10. Moreover, Tr1-like T cells were induced after culture with bone marrow−derived DCs and IL-10. A natural population of DCs with the same phenotype also exists in vivo. A single injection of immature DCs loaded with an influenza peptide led to the generation of cells that produced IL-10. A similar study showed that repeated injections of self-peptide−loaded immature bone marrow−derived DCs protected against subsequent induction of autoimmune disease, through the generation of IL-10−producing CD4+ T cells. Taken together, these studies imply that DCs are the most likely APCs for tolerance induction after soluble peptide administration
Duration of tolerance
The duration of tolerance induced by peptide therapy has not been studied extensively in humans, but preliminary data suggest that a single administration modifies the immune response to allergen for several months. Experimental models have yielded similar results, although in disease prevention rather than treatment protocols. The dominant epitope for autoimmune encephalomyelitis in the H-2u mouse is the N-terminal nonamer of MBP. Mucosal (intranasal) or systemic (intraperitoneal) administration of a soluble form of this peptide was effective in preventing autoimmune disease. Specific suppression of the response to the N-terminal peptide was virtually complete for 1−6 weeks after administration of a single dose. From 8 weeks onward, responsiveness slowly recovered in euthymic, but not adult, thymectomized mice. Such treatment substantially reduced mean maximal grades of disease, when disease was induced 1 week after treatment. But mice were barely protected from disease induction 16 weeks after peptide treatment. This observation suggests that T cell tolerance following peptide therapy is a thymus-independent, peripheral phenomenon, the reversal of which is dependent on new T cells being exported from the thymus. In practice, sustained suppression by peptide therapy will probably require repeated doses of suitably selected peptide antigens.
In conclusion, we believe the uncertainty surrounding 'specific' immunotherapy is diminishing as our understanding of immune regulation progresses. Investigators in the field are entering an era in which many of the questions surrounding this area of immunology will be addressed. These include clarification of the appropriate dose of antigen, the selection of peptide epitopes for induction of bystander suppression, the optimal route of administration for each specific disease and perhaps, the selection of adjuvants to enhance the therapeutic effect of soluble antigens.