The Prospect of Vaccination to Prevent Type 1 Diabetes

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United States. Diabetologia 2001; 44:354-62. 31. Green A, Patterson CC. Trends in the incidence of childhood-onset diabetes in Europe. 1989-1998.
[Human Vaccines 1:4, 143-150; July/August 2005]; ©2005 Landes Bioscience

The Prospect of Vaccination to Prevent Type 1 Diabetes Review

Received 04/05/05; Accepted 06/18/05

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INTRODUCTION

The maintenance of tolerance to self remains the central question in immunology. Paul Ehrlich, the father of immunology, recognised over 100 years ago that breakdown of immune tolerance to self might lead to ‘horror autotoxicus’. However, despite many subsequent advances in immunology, derived largely from mouse studies, the mechanisms underlying the breakdown of tolerance to self in human autoimmune diseases remain poorly understood. Mouse models demonstrate that developing lymphocytes may be deleted by apoptosis and purged from the immune repertoire if their receptors recognise with high avidity self-antigen peptides bound to major histocompatibility complex (MHC) molecules.1 This dogma of central tolerance holds that ‘horror autotoxicus’ is averted because self-reactive lymphocytes are deleted ab initio, but it is too facile a solution for what is manifestly a more complex problem. Thus, it is hardly possible to conceive of separate universes of self and nonself, of lymphocyte receptors that are not promiscuous and of antigen ligands or their mimics that are constrained by some metaphysical boundary between self and nonself, yet at the same time demand the specificity and diversity of cognition required of an efficient immune system. The simple, dichotomous self-nonself paradigm of central immune tolerance ignores the conservation of biological matter and form from microbes to mammals. Although self-antigens are expressed ectopically in the thymus and may serve to target and delete high avidity, potentially pathogenic T lymphocytes1,2 central tolerance is by no means fail-safe. T lymphocytes that react to these same self-antigens happily reside in the periphery of healthy individuals.3 Furthermore, it is now apparent that genes and proteins are subject to tissue-specific modifications such as alternate RNA splicing4 or post-translational covalent modifications5 that can insert antigenicity de novo into a post-thymic immune repertoire. Moreover, not all T lymphocytes that emerge from the thymus are necessarily pathogenic; cells with regulatory functions are also selected by self-antigens in the thymus.6 Evidence is also emerging that self-antigens are also ectopically expressed by a subset of myeloid lineage cells in the periphery,7 implying that the paradigm for central thymic tolerance may extend to the maintenance of peripheral tolerance. That physiological autoimmunity doesn’t usually progress to autoimmune disease attests to the importance of peripheral mechanisms of self-tolerance.8 First, self-antigens may be ignored by adaptive immune T and B lymphocytes in the absence of ‘danger’ signals, e.g., from microbes or tissue injury, needed to first activate innate immune and antigen-presenting dendritic cells.

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The author is supported by the National Health and Medical Research Council of Australia and by a Center Program Grant from the Juvenile Diabetes Research Foundation. He is indebted to his colleagues and students in clinical medicine and laboratory research, and to people and families with diabetes, for much support and inspiration. Catherine McLean provided excellent administrative assistance.

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ACKNOWLEDGEMENTS

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diabetes, Type 1, vaccine, autoantigen, virus, (pro)insulin, mucosa, T cell, tolerance, clinical trial

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Previously published as a Human Vaccines E-publication: http://www.landesbioscience.com/journals/vaccines/abstract.php?id=1923

Type 1 diabetes (T1D) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing β cells in the pancreatic islets. Primary prevention by traditional ‘positive’ vaccination awaits evidence that infectious agents trigger T1D. The pre-clinical phase of T1D, in which at-risk individuals can be infected by the presence of autoantibodies to islet anigens, is a window for secondary prevention. The Holy Grail of therapy is ‘negative’ vaccination to induce immune tolerance against disease-specific autoantigens that drive immune-mediated pathology. This can be achieved by administering autoantigen via a ‘tolergenic’ (e.g., muscosal, intradermal) route, cell (e.g., resting dendritic cell), mode (e.g., with blockade of c0-stimulation molecules) or form (as an ‘altered peptide ligand’). Although effective in rodent models of autoimmune disease, these strategies have so far been disappointing in humans. This review discusses the prospects of vaccination to prevent T1D, focusing on autoantigen-specific mucosal tolerance.

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Correspondence to: Leonard C. Harrison; Autoimmunity & Transplantation Division; The Walter & Eliza Hall Institute of Medical Research; 1G Royal Parade; Parkville 3050; Victoria, Australia; Tel.: 61.3.9345.2460; Fax: 61.3.9347.0852; Email: [email protected]

ABSTRACT

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Leonard C. Harrison

www.landesbioscience.com

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Second, following activation by antigen, lymphocytes may be deleted by apoptosis or rendered unresponsive (anergic) and/or induced to acquire regulatory properties, depending on the strength and nature of the signal they receive. A more laissez-faire view of the selfnonself question has emerged, post the static, centralist model. Tolerance, once described in almost moralistic metaphors (‘forbidden clone’, ‘original antigenic sin’), is now regarded as a property of a more complex, internally-regulated homeostatic system. Immunology imitating life! Nature deals a hand of immune genes shaped by evolution, mainly through encounter with microbes. Depending on our particular inherited gene set and how it is challenged post-natally we are, more or less, susceptible to breakdown of self-tolerance and autoimmune disease. This is illustrated by insulin-dependent or type 1 diabetes (T1D), an autoimmune disease in which innate and adaptive immune cells progressively destroy insulin-producing β cells in the islets of the pancreas. Human leukocyte antigen (HLA) genes in the major histocompatibility complex (MHC), specifically alleles at the HLA DR and DQ loci, are the single most important genetic determinant of T1D.9 Risk is highest with the HLA DR3,4; DQ2,8 haplotype and decreases, reflected by less aggressive pathology and a later age of clinical presentation, as the number of risk alleles carried decreases.10 The HLA DR2; DQ6 haplotype, on the other hand, confers protection against T1D. HLA molecules shape immune responses, good or bad, by binding specific antigenic peptides for recognition by T-cell receptors. HLA genetics accounts in large measure for why most of us don’t develop T1D. As well as non-HLA genes, environmental factors such as microbes and dietary components may promote or retard the development of T1D, as discussed below. There is little we can do about an inherited repertoire of potentially pathogenic lymphocytes except to prevent its activation or enhance its regulation in ways that mimic physiologic immunity. This is the conceptual framework on which the prospects for vaccination to prevent T1D will be examined.

PREVENTION OF T1D

Ideally, T1D would be eradicated by primary prevention aimed at avoiding or averting environmental factors thought to promote disease in genetically at-risk individuals, but these factors have not been clearly identified and may be ubiquitous. Without knowing what they are (and without being able to modify genetic susceptibility), the prospects for primary prevention remain uncertain. Secondary prevention, after the disease process has started, has been the focus of considerable attention since the early 1980’s, with many candidate agents, mainly immunosuppressive drugs, being trialled, usually after the onset of clinical diabetes (reviewed in ref. 11). Prevention is however more applicable to early, preclinical disease rather than to recent-onset clinical disease, when β-cell destruction is more advanced. Individuals in the preclinical phase of T1D can be identified by the presence of circulating autoantibodies to specific islet antigens: (pro) insulin, the mol. wt. 65,000 isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like insulinoma antigen 2 (IA2) (reviewed in ref. 12). The case for intervening early in these asymptomatic, at-risk individuals rests on the likelihood of greater efficacy, but demands careful consideration of safety (Fig. 1). Vaccines to promote protective immune homeostasis should be relatively safe and potentially more efficacious in the preclinical phase, whereas potentially toxic immunosuppressive drugs would require strong justification in asymptomatic individuals, especially children. In 144

Figure 1. Preventing type 1 diabetes: early versus late intervention.

Table 1

Markers of risk for diabetes in an islet autoantibody-positive relative



Number of antigen specificities of islet autoantibodies



Antigen specificity of islet autoantibody



Level of islet autoantibody



Age at detection of islet autoantibody



First phase insulin response (FPIR) to i.v. glucose



Insulin resistance, eg estimated as HOMA-R



HLA alleles for risk or protection



HLA haplotype sharing with proband



Kinship with proband

individuals with recent-onset disease, immunosuppressive drugs could be used to reduce the burden of pathogenic immunity and allow the emergence or active induction of protective immunity, with the potential for β-cell recovery and possible regeneration. Based on current knowledge of pathogenetic mechanisms, the outcomes of vaccination strategies to prevent T1D could be to: (1) avert environmental ‘trigger(s)’; (2) delete or inactivate pathogenic T cells; (3) induce protective/regulatory T cells; (4) promote β-cell protection; (5) enhance insulin action. A prerequisite for the development of a human therapeutic is the demonstration of efficacy and safety in animal models. The NOD mouse, the most widely used animal model of T1D, has contributed substantially to our understanding of disease mechanisms and the expectation that T1D is preventable.13 Autoimmune diabetes in the NOD mouse shares features with human T1D, including polygenic inheritance dominated by genes for antigen-presenting molecules in the MHC, autoimmune responses to (pro) insulin and GAD65, transfer of disease by bone marrow and a protracted preclinical phase.14 In contrast to humans, the NOD mouse is inbred and responds to many immune and other interventions.15 Nevertheless, most interventions prevent disease in only a proportion of NOD mice, some simply retard disease and others have no effect (and therefore are not reported), and many of the reported disease modifiers have not been tested in humans. In considering autoantigen-specific, ‘negative’ vaccination strategies for inducing immune tolerance, the NOD mouse has been the ‘proof-of-concept’ model.

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VACCINATION FOR WHOM? In Finland, which has the highest incidence of T1D in the world, neonatal screening for high-risk HLA class II susceptibility genes can identify over half of those destined to develop T1D.16 Neonatal screening to identify the genetically at-risk is a platform for primary prevention, but its modest predictive value would only justify an intervention that was safe for the whole population, for example a vaccine against a ubiquitous pathogen. When there is any question of safety, a prerequisite for intervention in asymptomatic individuals is the ability to predict development of clinical disease. Most studies of people at risk for T1D have centered on first-degree relatives of a T1D proband, who have been identified as positive for autoantibodies to one or more islet autoantigens, i.e., (pro)insulin, GAD and IA2. Risk markers in this population are well-documented12,17-19 (Table 1). In young, first-degree relatives with normal β-cell function, the 5-year risk of diabetes is of the order 50% if they have autoantibodies to one, two and three islet autoantigens, respectively. These predictions have been validated in the Diabetes Prevention Trial-1 (DPT-1) trials of systemic insulin20 and oral insulin.21 The addition of measures of insulin secretion and insulin action further refine risk. Thus, first phase insulin response (FPIR) to intravenous glucose below the 10th percentile signifies a poor prognosis20 and in autoantibody-positive relatives with a normal FPIR the highest risk is conferred by insulin resistance.22 The latter has important implications for preventative approaches directed at improving insulin action, as well as for selecting or stratifying autoantibody-positive subjects for prevention trials. First-degree relatives, with shared susceptibility genes and environmental risk factors, have at least a 10-fold higher overall prevalence of T1D than the background population but still they represent no more than 15% of people diagnosed with T1D. The risks in first-degree relatives cannot necessarily be extrapolated to the general population because the predictive value of a risk marker reflects the population prevalence of disease, according to Bayes’ theorem. The predictive value of islet autoantibodies in the general population has not been widely investigated but will be relevant once effective means of secondary prevention are found.

VACCINATION—EXOGENOUS ANTIGENS

Before discussing specific exogenous antigens, it should be mentioned that diabetes development can be suppressed in NOD mice by vaccination with ‘nonspecific’ immunostimulatory agents, including complete Freund’s adjuvant, bacillus Calmette-Guerin (BCG) and Schistosoma mansoni23,24 and DNA or CpG oligonucleotide.25 These agents stimulate innate immune pathways through toll-like and other pattern-recognition receptors and may reset immune homeostasis. Initial trials of BCG and Q fever vaccination in humans with recent-onset T1D have not demonstrated benefit to preserve residual β-cell function (reviewed in refs. 11, 26 and 27), but such nonspecific immune stimulation, discussed later in the context of the ‘hygiene hypothesis’, is unlikely to be particularly effective late in disease. One group has reported that vaccination of infants at birth with BCG is associated with a decreased risk of T1D,28 but this strategy has not been formally trialled. Primary prevention could be achieved by targeting environmental agents that promote islet autoimmunity. The evidence for these agents is largely circumstantial, but persuasive. In discordant identical twin pairs, the lifetime risk of diabetes for the nondiabetic twin is www.landesbioscience.com

50–70%;29,30 the incidence of T1D is increasing in many countries, particularly in the very young;31,32 in both hemispheres of the world a role for viruses is suggested by a peak in spring-summer births of children who will develop T1D33,34 and a peak in winter for the diagnosis of T1D;31,34,35 T1D has occurred after exposure to rubella virus in utero36 and to the β-cell toxin ‘Vacor’.37 Interest in environmental agents is centred mostly on dietary components and enteric viruses. Although we focus18 on promotion of disease by environmental agents, some agents may promote or protect depending on the circumstances. This applies particularly in the context of their interaction at the level of the mucosa and mucosal immune system. The T1D susceptibility HLA haplotype, A1-B8DR3-DQ2, which predisposes not only to T1D but also to celiac disease and selective IgA deficiency, is associated with increased immune responses to dietary proteins (reviewed in ref. 38). Individuals with this diabetogenic haplotype have evidence of impaired mucosal barrier function, with increased intestinal permeability.39 The requirement for a normal mucosa to maintain immune homeostasis is suggested by the effect of a germ-free versus conventional ‘dirty’ environment on diabetes incidence in NOD mice. The incidence of spontaneous diabetes in NOD mice differs greatly between colonies around the world and appears to be inversely correlated with exposure to microbial infection.40 An incidence of diabetes approaching 100% in mice housed under germ-free conditions can be significantly reduced by conventional conditions of housing and feeding.41,42 Under ‘dirty’ conditions, bacterial colonisation of the intestine leads to an increase in the number of differentiated intra-epithelial lymphocytes (IELs)43 and to maturation of mucosal immune function.44 IELs, at the interface between ‘self’ and ‘non-self’, are required for mucosa-mediated immune tolerance and for the generation of regulatory T cells in response to orally-administered antigen.45 One of these antigens could be insulin, a key autoantigen in T1D,46 which is present in breast milk.47 Apart from the influence of the microbial environment on maturation of mucosal immune function, animals in ‘dirty’ environments are exposed to a range of infections. How early microbial exposure relates to the acquisition of immune homeostasis remains unclear, but the rodent data support the hypothesis that the increase in prevalence of autoimmune and allergic disorders in the first world is due to ‘clean living’ conditions —the ‘hygiene hypothesis’.48,49

VIRUSES

Viruses could promote T1D in several possible nonexclusive ways: (1) direct infection of β cells; (2) infection of the exocrine pancreas with bystander death of β cells; (3) infection of the mucosa leading to increased intestinal permeability to diabetogenic dietary components or to loss of immune regulation and emergence of normally-suppressed diabetogenic T cells; (4) molecular mimicry between T-cell epitopes in a virus and in β-cell autoantigens; (5) superantigen stimulation of T cells. If a specific virus was clearly incriminated, vaccination of children early in life would be indicated, assuming that the vaccine was generally safe and did not elicit pathogenic molecular mimicry. Rubella. The first virus to be associated with T1D was rubella.36 Children with congenital rubella born to mothers who contracted rubella early in pregnancy had evidence of infection in the brain, pancreas and other tissues and approximately 20% developed insulin-dependent diabetes.50 Subsequently, almost twice this proportion of children with congenital rubella were reported to develop

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islet cell antibodies.51 Children with congenital rubella and ensuing diabetes were noted to have a higher frequency of the T1D susceptibility haplotype HLA-A1-B8-(DR3-DQ2).52 More recently, evidence for molecular mimicry between rubella and GAD peptides has been reported53 but the relevance of this is uncertain. The rubella vaccine has virtually eliminated congenital rubella and may represent the first example of primary prevention of T1D. Clearly though, additional environmental factors are involved because the incidence of T1D has continued to increase in “first world” countries. Despite the report of rubella-GAD mimicry,53 strengthened by the finding of islet autoantibodies in children infected with rubella virus,54 there is no evidence from multiple studies55,56 that the vaccination with attenuated rubella virus is associated with islet autoimmunity. Only 4% of girls receiving live attenuated rubella vaccine developed islet cell antibodies and these were transient and of low titre.57 Enteroviruses. Mothers infected during pregnancy with some enteroviruses, such as coxsackie and echoviruses, were reported to be more likely to deliver children who developed T1D early in life,58,59 but more recent studies have not confirmed this.60,61 Enterovirus RNA was isolated from peripheral blood mononuclear cells of 50% of recent-onset T1D cases in Scandinavia, and 26% of siblings and 0% of age- and sex-matched controls.62 In Germany60 and Australia (Honeyman MC, personal communication), coxsackie B virus infections were noted to be more common in infants and children with preclinical T1D than in controls but were not coincident with the detection of islet autoantibodies. Evidence for induction of T1D by mimicry with enteroviruses remains unconvincing.63,64 Because of their circumstantial association with T1D, enteroviruses remain a dubious vaccine target. Furthermore, there are 72 recognized serotypes and many thousands of enterovirus strain variants, and no enterovirus vaccines except for polio virus yet exist. However, if a diabetes-associated strain such as coxsackie B3, B4 or echo six were to be clearly implicated it would be a candidate vaccine for genetically at-risk children. Mumps. Epidemics of mumps virus have been associated with onset of T1D 2-4 years later.65,66 Intriguingly, the introduction of a mumps vaccine was associated with a plateau in the rising incidence of T1D in Finland,67 but this was temporary and mumps vaccination is unlikely to be of value for preventing T1D. Cytomegalovirus. Cytomegalovirus (CMV) can damage β cells68 and contains a peptide sequence mimic of a T-cell epitope in GAD recognized by a T-cell clone from a subject with Stiff Man Syndrome, a neurological disorder associated with autoantibodies to GAD.64 However, the evidence for CMV infection in T1D is weak.69 Furthermore, while CMV is a major cause of congenital defects, development of a vaccine is problematic because of latency and the carcinogenic potential of an attenuated virus. Rotaviruses. The discovery of strong sequence similarities between T-cell epitopes in IA2 and GAD and the VP7 protein of rotavirus in islet antibody-positive relatives70 suggested that molecular mimicry with rotavirus could precipitate islet autoimmunity. Rotavirus epidemics occur each winter particularly in kindergartens and are the most common cause of gastroenteritis in children. Rotavirus provides a profound inflammatory stimulus to the gut until sufficient IgA develops by about five years of age. In the Australian BabyDiab Study of genetically-at risk infants followed from birth, rotavirus infections were temporally associated with increases specifically in islet autoantibodies in 24 children before they developed diabetes.71 It was then shown that rotavirus could infect β cells in islets from mice, pigs and monkeys.72 Recently, it has 146

been found (Honeyman MC) that a majority of children, both at-risk for T1D and HLA-matched controls, have T-cell responses to the similar peptide sequences in rotavirus VP7, and IA2 and GAD. Thus, while ubiquitous rotavirus infections may drive cross-reactive immunity to islet autoantigens this alone is not diabetogenic. Mimicry could, however, complement and sustain the immune response to direct infection of β cells. It is also necessary to consider that the outcomes of enteric infection in infants may be complex, e.g., depending on timing in relation to mucosal development and breastfeeding. Infection by rotavirus or other potentially diabetogenic viruses in an anti-inflammatory context, such as during breast feeding, could conceivably be protective against the development of T1D. Rotavirus vaccines have been developed but were withdrawn because of safety concerns following cases of intestinal intussusception. Whether a vaccine to rotavirus or any other potentially diabetogenic virus would alter the incidence of T1D is unanswerable without vaccination of the population at large. T1D might be eradicated if just one virus was responsible for most cases, but it seems more likely that the environmental agents will be ubiquitous.

VACCINATION—ENDOGENOUS ANTIGENS

The prevention strategy that comes closest to the ideal for preclinical disease, or even primary prevention, is so-called ‘negative’ vaccination against the autoantigen(s) that drive β-cell pathology, to induce disease-specific immune tolerance. This strategy is based on the concept that self-antigen-specific immunoregulatory mechanisms are physiological and can be boosted or restored to prevent pathological autoimmunity. Ways of achieving this include the administration of autoantigen by a ‘tolerogenic’ route (mucosal), cell type (resting dendritic cell), mode (with blockade of costimulation molecules) or form (as an ‘altered peptide ligand’), all of which can suppress the development of experimental autoimmune diseases in rodents.73-75 The mechanisms include deletion of and/or induction of anergy in potentially pathogenic T cells and induction of regulatory T cells (Treg). Some Treg secrete anti-inflammatory cytokines such as IL-10 or TGF-β that may then impair the ability of dendritic cells to elicit T helper 1 (Th1) or cytotoxic T-cell responses to any antigen locally at the site of the lesion or in the draining lymph nodes, a phenomenon called ‘bystander suppression’. Several autoantigens are associated with T1D,11 but their place in the hierarchy of drivers of β-cell destruction remains problematic.’Bystander suppression’ obviates the need to know if the autoantigen used to induce tolerance is necessarily the major or primary pathogenic autoantigen.73-76 In the case of T1D, a considerable body of evidence indicates that proinsulin, the only β cell-specific autoantigen, plays a preeminent role in driving autoimmune β-cell destruction.

MUCOSA-MEDIATED VACCINATION: PROOF-OF-CONCEPT IN THE NOD MOUSE

Many experiments have shown that it is possible to partially protect NOD mice from spontaneous diabetes by the mucosal administration of islet autoantigen. Most studies have been performed with insulin or with peptides from proinsulin. Initially, Zhang et al.77 reported protection after oral porcine insulin. Bergerot et al78 then showed that human insulin induced CD4+ Treg that could transfer protection to naïve mice. Protection following oral insulin was reported to be associated with decreased expression of IFN-γ-secreting

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Th1 T cells in the pancreas and pancreatic lymph nodes.79,80 Oral insulin-induced CD4 Treg have also been shown to prevent immunemediated diabetes induced by lymphocytic choriomeningitis virus (LCMV) infection of mice expressing the viral nucleoprotein of LCMV under control of the rat insulin promoter in their β cells.81 The majority of T cells in the islets of oral insulin-treated mice without diabetes secreted the Th2 (IL-4, IL-10) and Th3 (TGF-β) cytokines, in contrast to IFN-γ-secreting Th1 cells in islets of mice developing diabetes. The protective effect of oral insulin was enhanced by simultaneous feeding with IL-10,82 bacterial component OM-8983,84 or Schistosome egg antigen,85 all of which promote Th2 responses. Fusion of insulin to cholera toxin B-subunit (CTB) significantly improved the ability of oral insulin to prevent diabetes.86 Oral CTB-insulin conjugates in NOD mice induced a shift from a Th1 to a Th2 immunity associated with the induction of regulatory CD4 T cells.87 NOD mice were protected from diabetes by feeding potatoes that transgenically express CTB-insulin conjugates.88 Oral GAD was also shown to suppress diabetes development in NOD mice.89 Although it is generally believed that neonates are less susceptible to mucosal tolerance induction, oral administration of insulin, insulin B-chain or GAD peptide during the neonatal period still suppressed diabetes development in NOD mice.90 This suggests that even in very young infants delivery of autoantigen to the mucosa, eg insulin in milk, could be prophylactic. Protection against diabetes in NOD mice can also be achieved by naso-respiratory administration of islet autoantigens. This route of direct delivery to the mucosa avoids antigen degradation. When insulin was given as an aerosol to NOD mice at 8 weeks of age, after the onset of subclinical disease, insulitis and diabetes incidence were both significantly reduced.91 Aerosol insulin induced novel anti-diabetic CD8 γδ T cells that suppressed the adoptive transfer of diabetes to nondiabetic mice by T cells of diabetic mice. The type of Treg induced by mucosal administration of (pro) insulin depends on the route and form of antigen. Naso-respiratory insulin, nondegraded and conformationally intact, induces CD8 γδ Treg, whereas oral insulin degraded to peptides, or intranasal or oral (pro) insulin peptides, induce CD4 Treg.92,93 Intranasal administration of the insulin B chain peptide (aa9-23), an epitope recognised by islet-infiltrating CD4 T-cell clones capable of adoptively transferring diabetes to naïve mice, induced CD4 Treg and protected NOD mice from diabetes.94 A peptide that spans the B-C chain junction in proinsulin also induced CD4 Treg after intranasal administration.93 This peptide, like insulin B9-23, binds to the NOD mouse class II MHC, I-A g795 and is a T-cell epitope in NOD mice96 and humans at risk for T1D.97 T-cell epitope peptides from GAD administered intranasally were also protective, being associated with induction of regulatory CD4 Treg and reduced IFN-γ responses to GAD.98 Islet autoantigen proteins or peptides that induce Treg are potential ‘vaccines’ for intranasal delivery to prevent T1D in humans, but this promise remains to be fulfilled (see below).

TRIALS OF ISLET AUTOANTIGEN-SPECIFIC VACCINATION IN HUMANS

The large multi-centre Diabetes Prevention Trial 1 (DPT-1) was launched in the United States in 1994 to determine whether autoantigen-specific therapy with either systemic or oral insulin would delay or prevent the onset of diabetes in at-risk relatives. Previously, intensive systemic insulin therapy had been reported to prolong the ‘honeymoon phase’ after diagnosis99 and a pilot study of www.landesbioscience.com

prophylactic systemic insulin had suggested that this approach might be of benefit in delaying the onset of diabetes in at-risk relatives.100 It remains unclear though whether systemic insulin would have acted only as a hormone to control blood glucose and ‘rest’ the β cells but also as an antigen to induce immune tolerance presumably by activating deletion of insulin-specific T cells. In DPT-1, low dose systemic insulin (annual intravenous insulin infusions and daily subcutaneous injections) was given to a high-risk group of relatives (>50% risk of diabetes over five years), matched with an untreated but closely monitored control group. Unfortunately, this treatment had no effect on diabetes incidence.20 The more recent randomised control DPT-1 of oral insulin recruited relatives with a 26–50% five-year risk of developing diabetes, but again the primary analysis revealed no effect.21 Two trials of oral insulin (up to 7.5 mg daily for 12 months) in recently-diagnosed patients showed no protective effect on residual β-cell function.101,102 There are probably several reasons why these trials failed: in some cases, selection of subjects with end-stage disease; inadequate dose or bioavailability of the agent, possibly related to the route of administration; coinduction of pathogenic T cells. Apart from the fact that the ‘damage has been done’ and may not be reversible, induction of antigen-specific immunoregulation alone without concomitant inactivation or deletion of pathogenic effector cells is likely to be relatively ineffective in late preclinical disease. If a balance between pathogenic and protective T cells determines clinical outcome, then antigen-specific immunoregulation should be most effective in early preclinical disease (Fig. 1). Regarding route of administration, oral delivery may not be optimal for mucosa-mediated tolerance, because proteins are generally degraded after ingestion, whereas even in the case of a small peptide responses may be seen after naso-respiratory but not oral delivery.103 In the mouse, at least, the nose seems to be an efficient route for activating mucosa-associated lymphoid tissues. Thus, nasal delivery of the model antigen, ovalbumin, elicited antigen-specific T-cell responses in cervical, mediastinal and mesenteric mucosal lymph nodes, whereas oral delivery elicited responses only in the mesenteric nodes.104 However, irrespective of route, mucosal delivery of an antigen can be a ‘double-edged sword. Nasal, aerosol or oral ovalbumin was associated not only with classical mucosal tolerance but with induction of pathogenic CD8+ cytotoxic T cells that could destroy β cells expressing transgenic ovalbumin.105 To achieve a clinical effect from tolerance induction, it was necessary to block induction of pathogenic T cells by transient coadministration of systemic anti-CD40 ligand monoclonal antibody.105 Whether mucosally-administered insulin is also a ‘double-edged’ sword is unknown. However, a proinsulin B-C chain peptide (aa B24-C36) that induces CD4+ Treg in NOD mice after nasal delivery is a ‘combitope’ of CD4+ and CD8+ T-cell epitopes and is protective only if the C-terminal p9 anchor residue for binding to MHC class I (Kd) is either deleted or mutated.93 Failing this, protection can be afforded, as described for ovalbumin105 by blockade of CD40 ligand (Martinez NR, Harrison LC, submitted). These findings further underscore the necessity to evaluate immune responses to mucosal autoantigens in human trials. The rationale for the DPT-1 trial of oral insulin was the induction of ‘oral tolerance’ in the NOD mouse. In the mouse, milligrams of gavaged insulin were required to induce ‘regulatory’ anti-diabetogenic CD4+ T cells and partially suppress development of diabetes.77,78 Yet, the daily dose of insulin given orally in the human trials was only 7.5 mg which, on a body weight basis, equates to only a few micrograms in the mouse. Oral insulin is degraded and its bioavailability to

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induce mucosa-mediated immune tolerance in the upper small intestine is unpredictable. Despite this, dose-ranging studies in humans to determine a bioavailable dose of oral insulin were not undertaken. Insulin antibodies as a readout of bioavailability were observed after aerosol insulin in NOD mice91 and after intranasal insulin in humans.106 Irrespective of whether such antibodies are a marker of immunoprotection, they demonstrate that the dose used was bioactive. Insulin autoantibodies are a risk marker for T1D and therefore the increase in insulin antibodies after administration of naso-respiratory insulin would seem paradoxical with immunoprotection. However, this increase in humoral immunity was accompanied by a decrease in T-cell proliferation to insulin after intranasal insulin,106 entirely consistent with the concept of mucosal tolerance. Reciprocal cellular and humoral immune responses, originally termed ‘immune deviation’ by Parish,107 and later popularised in terms of the Th1/Th2 paradigm, are a feature of the earliest descriptions of mucosal tolerance, confirmed in landmark but overlooked human studies with the experimental antigen, keyhole limpet hemocyanin (KLH). When KLH was administered orally108 or nasally109 to human volunteers, subsequent antibody and T-cell responses to systemic immunization with KLH increased and decreased, respectively. Enthusiasm to translate mucosal tolerance from rodents to humans has been tempered by failure to show clinical benefit not only of oral insulin but also of oral myelin basic protein in multiple sclerosis110 and oral collagen in rheumatoid arthritis.111,112 As suggested, these failures could be due to an inability of mucosal tolerance to counteract pathogenic T cells in end-stage autoimmune disease or to the coinduction a pathogenic immune response. In addition, as with the oral insulin trials, none of these trials reported an immune effect of the oral antigen. The question must be raised whether such major trials are judicious without reassurance that the dose of antigen used is bioavailable and has an immune effect. At the same time, there is an urgent need for the measurement of potentially relevant bio-markers, particularly pro-inflammatory (potentially pathogenic) and anti-inflammatory (regulatory) T cells, to be standardised and incorporated into trials. The Melbourne intranasal insulin trial I (INIT I)106 was a randomized controlled crossover pilot trial of intranasal insulin vaccine in young T1D relatives (median age 10.8 yr; n = 38) with islet autoantibodies. Two 400 µg doses of insulin per nostril were self-administered daily for ten days, then on two consecutive days each weekend, for six months. The aim was to determine if intranasal insulin was safe and would induce changes in surrogate immune and metabolic markers consistent with an immunoprotective effect. No local or systemic adverse effects were observed. Diabetes developed in 12 subjects who had negligible β-cell function at entry, after a median of 1.1 years. β-cell function in the remaining 26, the majority of whom had antibodies to two or three islet autoantigens and FPIR > first percentile at entry, generally remained stable over a median follow-up of 3.0 years. Intranasal insulin was associated with an increase in anti-insulin antibody and a decrease in T-cell responses to (denatured) insulin. This trial identified a dose of intranasal insulin that was safe and which induced changes in immunity to insulin as previously reported in NOD mice. Because it was a crossover trial in which all subjects received treatment with intranasal insulin for six months, it could not determine if intranasal insulin prevents loss of β-cell function and diabetes. This will be answered in a follow-up trial. In adults with autoimmune diabetes there has been only one published clinical trial of prophylactic insulin. This small study by 148

Kobayashi et al.113 demonstrated that low doses of subcutaneous insulin improved the C-peptide response to oral glucose and decreased HbA1c at 6 and 12 months after diagnosis, compared to treatment with oral sulfonylurea drugs. Whether the beneficial effect of insulin treatment was due to metabolic or immunoregulatory mechanisms is not known. A randomised controlled Phase 2 trial of intranasal insulin (INIT III) in adults with recent-onset autoimmune diabetes is nearing completion in Melbourne. A Phase 2 trial of subcutaneous GAD, sponsored by Diamyd Medical AB, is also underway in adults with recent-onset T1D.

HEMATOPOIETIC STEM CELL-MEDIATED VACCINATION

Allogeneic- or mixed-allogeneic bone marrow transplantation strategies are being trialled for severe autoimmune diseases,114 but are unlikely to be suitable for T1D due to the requirement for cytotoxic conditioning of the host, and the risks of graft rejection115 and graft-versus-host disease.116 A much safer approach would be to use autologous, genetically-engineered hematopoietic stem cells or their antigen-presenting cell progeny to introduce molecules into the hematopoietic compartment that could prevent autoimmune disease. Adopting this approach, we found that transfer to young, irradiated NOD mice of 103 syngeneic hematopoietic stem cells encoding proinsulin expression in antigen-presenting cell progeny totally prevented diabetes.117 This dramatic effect appears to depend on proinsulin expression by ‘resting’ immature dendritic cells.118 The application of this ‘cell therapy’ to humans faces two obstacles related to safety—introducing genes into stem cells without the risk of oncogenesis and avoiding toxic conditioning regimens in the host.

CONCLUSION

Prevention of T1D will probably occur incrementally through different interventions suited to risk level-disease stage and genotype. Environmental agents that promote autoimmune β-cell destruction are likely to be ubiquitous and therefore a single intervention, for example vaccination against a specific virus, is unlikely to be the ultimate solution. Autoantigen-specific vaccination applied rationally to enhance natural immunoregulatory mechanisms of protection is a safe and widely-applicable approach even to primary prevention, but its potential remains to be scientifically evaluated in humans. In end-stage disease, combinatorial approaches that not only enhance immune regulation but suppress pathogenic immunity are more likely to succeed. Advances in manipulating gene expression safely in stem cells and conditioning the recipient immune system should open new avenues for autoantigen-specific vaccination. Prevention of T1D will facilitate replacement or regeneration of β cells in people with established diabetes. Furthermore, the lessons learnt from the prevention of T1D should apply to other autoimmune diseases. References 1. Ohashi PS. Negative selection and autoimmunity. Curr Opin Immunol 2003; 15:668-76. 2. Derbinski J, Schulte A, Kyewski B, Klein L. Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self. Nat Immunol 2001; 2:1032-9. 3. Mannering SI, Morris JS, Jensen KP, Purcell AW, Honeyman MC, van Endert PM, Harrison LC. A sensitive method for detecting proliferation of rare autoantigen-specific human T cells. J Immunol Methods 2003; 283:173-83. 4. Garcia-Blanco MA, Baraniak AP, Lasda EL. Alternative splicing in disease and therapy. Nat Biotechnol 2004; 22:535-45. 5. Doyle HA, Mamula MJ. Post-translational protein modifications in antigen recognition and autoimmunity. Trends Immunol 2001; 22:443-9. 6. Caton AJ, Cozzo C, Larkin Jr, Lerman MA, Boesteanu A, Jordan MS. CD4(+) CD25(+) regulatory T cell selection. Ann NY Acad Sci 2004; 1029:101-14.

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Human Vaccines

2005; Vol. 1 Issue 4