A safer vaccine for Alzheimer’s disease?

Neurobiology of Aging 23 (2002) 1001–1008

A safer vaccine for Alzheimer’s disease? Einar M. Sigurdsson a,b,∗ , Thomas Wisniewski a,b,c , Blas Frangione a,b a

Department of Psychiatry, School of Medicine, New York University, 550 First Avenue, New York, NY 10016, USA Department of Pathology, School of Medicine, New York University, 550 First Avenue, New York, NY 10016, USA c Department of Neurology, School of Medicine, New York University, 550 First Avenue, New York, NY 10016, USA b

Received 12 April 2001; received in revised form 6 February 2002; accepted 21 February 2002

Abstract Recent reports indicate that amyloid-␤ (A␤) vaccine-based therapy for Alzheimer’s disease (AD) may be on the horizon. There are, however, concerns about the safety of this approach. Immunization with A␤1–42 may not be appropriate in humans because it crosses the blood–brain barrier, can seed fibril formation, and is highly fibrillogenic. A␤1–42 fibrils can in turn cause inflammation and neurotoxicity. This issue is of a particular concern in the elderly who often do not mount an adequate immune response to vaccines. Our findings show that vaccination with nonamyloidogenic/nontoxic A␤ derivative may be a safer therapeutic approach to impede the progression of A␤-related histopathology in AD. Although the site of action of the anti-A␤ antibodies has been suggested to be within the brain, peripheral clearance of A␤ may have a greater role in reducing cerebral amyloid plaques in these animals and eventually in AD patients. Antibodies in general are predominantly found outside the central nervous system (CNS) and will, therefore, primarily clear systemic A␤ compared to brain A␤. This disruption of the equilibrium between central and peripheral A␤ should then result in efflux of A␤ out of the brain, and subsequent removal of plaques. A␤ therapy can be targeted to the periphery, which may result in fewer CNS side effects, such as inflammation. Future A␤ derived vaccines should include Th epitopes, carriers and/or lipid moieties to enhance antibody production in the elderly, the population predominantly affected by AD. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Amyloid-␤; Alzheimer’s disease; Central nervous system; Therapy; Immunization

1. Introduction

2. Vaccination as therapy for AD

Evidence that amyloid may play an important role in the early pathogenesis of AD comes from various studies [11]: (A) genetic analysis of families with hereditary AD has revealed mutations in the gene for the amyloid-␤ precursor protein (APP), near or within the A␤ sequence (Fig. 1), in addition to mutations within the presenilin 1 and 2 genes. Most of these mutations lead to an increased production of A␤1–42 and/or total A␤, whereas some enhance the fibrillogenicity of A␤; (B) Down’s syndrome patients have three copies of the gene for the amyloid precursor protein and develop AD neuropathology at an early age; (C) A␤ protofibrils/fibrils are toxic in neuronal culture [48,54] and to some extent when injected into animal brains [38,39], whereas the soluble form appears to be less toxic [31]; and (D) inheritance of the apoE allele ε4 increases the risk of AD and correlates with increased cerebral amyloid deposition.

Currently, the only approved therapy for AD is cholinergic drugs that have minimal efficacy [15]. Other potential therapies include cholesterol-lowering drugs, anti-inflammatory compounds, hormonal therapies, anti-oxidants, metal complexing agents, compounds that reduce or inhibit A␤ synthesis or increase A␤ degradation, anti-␤-sheet conformational agents and passive/active immunization against A␤. Presently, compounds targeting A␤ hold the most promise as therapy for AD although less specific approaches affecting downstream events in the pathogenesis are likely to be used as supplementary treatments. We have reported on ␤-sheet breaker peptides that have a sequence similarity with the central hydrophobic region of A␤ but contain a proline, a ␤-sheet breaker amino acid. These peptides interfere with fibril formation and induce fibril disassembly both in vitro and in vivo [40,45,46]. Numerous additional studies have been performed in vitro on short A␤ derivatives and/or other A␤ binding agents with similar properties [6]. These compounds include monoclonal antibodies raised against the N-terminal region of A␤ that

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Corresponding author. Tel.: +1-212-263-7527; fax: +1-212-263-6751. E-mail address: [email protected] (E.M. Sigurdsson).

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Fig. 1. The A␤ molecule within the amyloid precursor protein is partially embedded within the cell membrane (residues 29–42). Mutations (brown color) in regions where the ␤- and ␥-secretases cleave lead to an increase in A␤1–40 and/or A␤1–42. Mutations within the A␤ sequence enhance its fibrillogenicity. The most widely used Tg mice (Tg2576) contain the Swedish double mutation. K6A␤1–30-NH2 contains the first 30 amino acids of A␤1–42, and thereby the two major antigenic sites of the A␤, amino acids 1–11 and 22–28. Six lysine molecules were added to the N-terminus to decrease fibrillogenicity and to possibly increase immunogenicity. The compound is amidated to maintain the antigenicity of the C-terminus.

disaggregate A␤ fibrils, maintain A␤ solubility and prevent A␤ toxicity in cell culture [7,8,43,44]. Schenk et al. [36] extended these studies to an in vivo situation by vaccinating Tg APP mice (PDAPP; APPV717F) with aggregated/fibrillar A␤1–42. These mice were immunized before the onset of AD-type neuropathology (at 6 weeks of age), or

when some amyloid deposition has occurred (at 11 months of age). At 13 months, the former group had virtually no amyloid plaques or associated histopathology. In the latter group, amyloid burden, neuritic dystrophy and astrogliosis was also significantly reduced in the A␤1–42 treated group both following 4 and 7 months treatment. The importance

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of anti-A␤ antibodies in diminishing plaques was subsequently confirmed by Bard et al. [2], where peripherally administered antibodies against A␤ were shown to enter the CNS and reduce brain parenchymal amyloid burden in the same mouse model. Following these reports, Weiner et al. [50] demonstrated that intranasal immunization with freshly solubilized A␤1–40 reduced cerebral amyloid burden in the PDAPP mouse. Then, two studies indicated that a vaccination-induced reduction in brain amyloid deposits resulted in cognitive improvements [17,28]. Both groups used a similar protocol as Schenk et al., which is injection of aggregated/fibrillar A␤1–42 peptide mixed with Freund’s adjuvant. Most recently, DeMattos et al. [4] demonstrated that antibody entry into the brain was not necessary for plaque removal, and we reported that soluble A␤ derivative can be used as an immunogen, indicating that antibody mediated clearance of soluble A␤, from outside the brain, may be important for plaque removal [41].

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can also cross the BBB and co-deposit on existing amyloid plaques leading to increased toxicity, and may actually promote plaque formation. These detrimental effects may go undetected in short-term clinical studies because the toxicity of A␤1–42 is likely to be chronic. AD is commonly thought to progress for decades before cognitive impairments are seen. Whether this occurs in Tg mice following A␤1–42 vaccination has not been investigated thoroughly. Presumably, none of the A␤1–42 vaccination studies reported to date have enough statistical power to observe this effect, because no data has been provided on individual correlation between titer and amyloid burden in these mice. A phase II clinical vaccination trial in which A␤1–42 was injected into individuals with AD was recently terminated because of cerebral inflammation observed in some patients. These side effects may be related to A␤1–42 toxicity and/or cell-mediated auto-immunity. Tailoring of the A␤ molecule as in our approach may eliminate these adverse reactions, while maintaining humoral immune response.

3. Safety issues 4. Non-toxic A␤ derivatives as immunogens An issue of concern regarding the safety of A␤-targeted vaccination approach is autoimmunity, which may occur in humans, although it has not been reported in vaccinated APP mice. Autoimmunity is an acquired immune reactivity to self-antigens, and autoimmune diseases occur when these responses lead to tissue damage. Differences of immune response to self-antigens in mice versus humans may be significant [49]. In addition to having high levels of human APP, the transgenic mice also express mouse APP at endogenous levels. The human APP and its A␤ derivative have a different amino acid sequence than their mouse counterparts, and in the study by Schenk et al. [36] the mice immunized with human A␤1–42 had as expected much higher titer against human A␤ than against mouse A␤. Hence, the mice may be less prone to developing an autoimmune disease than humans who only express human A␤. There are additional safety concerns specifically with the use of A␤1–42 in vaccine preparations for human use. First, A␤1–42 forms inflammatory/toxic fibrils [52], and it has been demonstrated by others and us [19–34,37,56], that A␤1–40/42 can cross the blood–brain barrier (BBB) in experimental animals. Also, even in small quantities A␤1–42 may seed fibril formation [18]. As reported by Kane et al. [20], intracerebral infusion of A␤-rich AD homogenate in Tg APP mice does result in the development of profuse A␤ plaques and vascular deposits 5 months following infusion whereas at 4 weeks no deposits were observed, and control Tg mice had only limited deposition of diffuse A␤ at the later timepoint. This safety issue is of a particular concern in the elderly who often do not mount an adequate immune response to vaccines. In these individuals with low antibody titers it is conceivable that aggregated/fibrillar A␤1–42, which was used for immunization by Schenk et al. can initiate and/or enhance congophilic angiopathy. A␤1–42

In the absence of prior knowledge of whether A␤1–42 will clear plaques in humans or if it will serve as a nidus for plaque formation, it is safer to use non-toxic immunogenic A␤ derivatives. We have designed a synthetic non-amyloidogenic peptide homologous to A␤ that has a reduced ability to adopt a ␤-sheet conformation and, therefore, confers a much lower risk of toxicity in humans (Fig. 1). This peptide was designed to have reduced fibrillogenic potential and enhanced immunogenicity while maintaining the major immunogenic sites of A␤ peptides, which are residues 1–11 and 22–28 [16]. Accordingly, the peptide contains the first 30 amino acid residues of A␤ with 6 lysine residues at the N-terminus. Poly-l-lysine enhances immunogenicity [51], and the coupling of lysine residues to the C-terminus of short A␤ sequences within the 15–25 domain of A␤ has recently been proposed by Pallitto et al. [30] in the design of anti-␤-sheet peptides or A␤ fibrillogenesis inhibitors. The use of potential peptide inhibitors of fibril formation as immunogens has never been previously proposed. Prior to vaccinating mice, we demonstrated that K6A␤1–30-NH2 has low ␤-sheet content, that it does not form fibrils, and is not toxic in human cell culture [41]. Subsequently, our immunization in Tg APP mice [Tg2576 [13]; APP695(K670N + M671L)] for 7 months with K6A␤1–30-NH2 , a non-amyloidogenic, non-toxic A␤ homologous peptide, reduced cortical and hippocampal brain amyloid burden by 89 and 81%, respectively [41]. Concurrently, brain levels of soluble A␤1–42 were reduced by 57% (Figs. 2–3). In other words, the use of this non-toxic A␤ derivative results in a similar reduction in amyloid burden as observed with the potentially toxic A␤1–42 [36]. Ramified microglia expressing interleukin-1␤ associated with the A␤ plaques were absent in the immunized mice indicating

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Fig. 2. Coronal sections (50×; original magnification) stained with 6E10 against A␤ (dark spots), through the hippocampus and cortex in a Tg control-(A) and K6A␤1–30-treated (B) Tg mouse. C and D are adjacent sections (100×) double stained for interleukin-1␤ (black) that recognizes microglia, and A␤ (red originally, see arrows). Note the reduction of amyloid burden in the immunized mouse (B), and the lack of ramified microglia (D) surrounding A␤ plaque in the same mouse, compared to a control mouse (A, C). The bars in A and C are 100 ␮m. Abbreviations: hip, hippocampus; cx, cortex; cc, corpus callosum. Reproduced from [41] with publisher’s consent.

reduced inflammation in these animals (Fig. 2). These promising findings, which we have reported previously [41], suggest that immunization with non-amyloidogenic A␤ derivatives represents a potentially safer therapeutic approach to reduce amyloid burden in AD, instead of using toxic A␤ fibrils.

5. Mechanism of plaque clearance The mechanism of the vaccination-induced reduction in cerebral amyloid burden is not fully understood. However, based on the passive vaccination study by Bard et al. [2], antibodies have a pivotal role, although it is questionable if the minute amount of antibodies that enter the brain are sufficient to result in plaque removal. It is, however, obvious that antibodies lead to plaque clearance if applied intracerebrally [1]. Differences in permeability of albumin and insulin have been reported in double Tg APP/presenilin 1 mice compared to non-Tg mice [35], and the A␤-antibody complex in non-Tg mice may be delivered across the BBB by the A␤ receptor-mediated transport [32]. There are no reports on the integrity of the BBB towards antibodies in Tg mouse models for cerebral amyloidosis, and it will be important to determine if the penetration of antibodies through the BBB in these animals is abnormally high because of some underlying pathology such as inflammation or if it is sim-

ilar to that in control non-Tg mice. Even though the access of antibodies into the CNS in these Tg mice may prove to be much greater than in control mice, it may still model the integrity of the BBB in AD, which is believed to be compromised [55]. It is unlikely that these antibodies are affecting the production of A␤ because they have been reported not to recognize APP [50]. The antibodies are likely to have their effect by enhancing clearance of A␤ by a variety of mechanisms. One pathway may be via microglial activation following antibody binding to A␤ plaques [2,36]. Their effect may also be in part caused by binding to soluble A␤ within the brain that alters the equilibrium between deposited A␤ versus soluble A␤, resulting in enhanced clearance of deposited A␤. A major pathway in the clearance of cerebral A␤ may be the binding of antibodies to soluble A␤ in peripheral fluids. The subsequent reduction in peripheral A␤ levels alters the equilibrium between A␤ found within and outside the CNS that may result in efflux of A␤ out of the CNS (Fig. 4). This possibility, which we put forward in our recent report [41], is supported by the fact that most antibodies are circulating in peripheral tissues and the Tg2576 mice have high levels of A␤ not only within the CNS but also in the periphery. Furthermore, antibodies to non-fibrillar A␤ derivatives such as ours may have higher affinity for soluble A␤, than those generated against fibrillar A␤. It has been suggested that the effect of immunization with aged A␤1–42 is via the generation of antibodies specific for fibrillar A␤. However,

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Fig. 3. Reduction in cortical (A) and hippocampal (B) amyloid burden (6E10) following 7 months treatment with K6A␤1–30-NH2 . There is an 89% reduction in cortical amyloid burden (∗ P = 0.0002; t-test; n = 4 per group) and an 81% reduction in hippocampal amyloid burden (∗ P = 0.0001). Soluble A␤1–42 levels (C) are reduced by 57% within the brains of the vaccinated mice (∗ P = 0.0019). Reproduced from [41] with publisher’s consent.

the successful generation of specific antibodies against amyloid ␤-sheet fibrils is rare. This type of conformational anti-amyloid antibody has been described for the scrapie form of the prion protein [22], and light chain-associated amyloid fibrils [12], but are not yet available for thorough analysis of their properties. Recent reports show that in the Tg2576 mice, plasma levels of A␤ decrease as cerebral plaque burden increases [21,23]. This suggests an interaction between these two compartments that can be manipulated, and is in agreement with our view that systemic A␤ may contribute to plaque formation within the brain [3]. Monitoring of plasma levels of A␤ throughout the immunization procedure in addition to comparing the plasma half-life of A␤1–40/42 in immunized versus non-immunized mice may give some

insight into this important issue. The clearance of A␤ from the circulation may be altered if soluble plasma A␤ is to some extent bound to antibodies in the immunized mice. This data, together with antibody affinity/avidity for soluble versus fibrillar A␤, the BBB permeability of A␤ antibodies and their binding motif (soluble, amorphous or fibrillar A␤), will clarify possible interactions between antibodies and A␤ within the CNS and in the peripheral system that may affect amyloid burden within the brain. These experiments will address the feasibility of targeting A␤ peripherally to reduce amyloid burden within the brain, which may result in therapy with fewer side effects because CNS entry will not be needed. Antibodies acting within the CNS may lead to detrimental microglia-related inflammation within the brain. Peripheral antibodies drawing out cerebral A␤

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Fig. 4. A hypothetical scenario underscoring the importance of the peripheral system in clearance of A␤ plaques within the brain. (A) In AD patients or Tg APP mice, it is not clear if equilibrium exists between soluble A␤ (sA␤) within the brain and in blood, although plaque formation is favored. (B) Following immunization, most of the antibodies (presumably >99.9%) are circulating within the bloodstream, and bound A␤ is cleared. Subsequent reduction in free sA␤ within the circulation results in efflux of sA␤ from the brain, which in turn shifts the equilibrium from plaque A␤ to sA␤. The overall effect is a prevention of plaque formation and/or a decrease in plaque size. BBB: blood–brain barrier.

through shift in equilibrium of soluble A␤ outside and within the CNS cannot produce this adverse effect. The importance of peripherally-mediated A␤ clearance which we suggested [41], was emphasized by the studies of DeMattos et al. [4]. They used the PDAPP Tg model that expresses human APP predominantly within the CNS [9]. Following i.v. injection of anti-A␤ antibody, plasma- and cerebrospinal fluid levels of A␤ increased acutely whereas no antibodies were observed labeling the plaques within the brain. The overall effect of the peripheral administration of the anti-A␤ antibody was to reduce amyloid plaque burden within the brain, presumably by increasing efflux of A␤ peptide out of the brain. These findings need to be confirmed in the more widely used Tg2576 model [13], which expresses human APP at high levels both within the CNS and in peripheral organs. Furthermore, passive immunization with anti-A␤ IgM may clarify the mechanism of antibody-mediated A␤ removal. IgM is composed of five four chain units whereas IgG contains only one four chain unit, and because of its size IgM should cross the BBB to a much lesser extent than IgG. In human cerebrospinal fluid, IgM is either undetectable [47] or found at levels up to few hundred-fold less than IgG [29]. Humans express APP at much lower levels

than the Tg mice and, therefore, have lower circulating levels of A␤. Monitoring of plasma A␤ levels in ongoing clinical safety studies on A␤ immunization will be useful to determine if a similar phenomenon may occur in humans.

6. Active versus passive immunization The most obvious advantages of passive immunization is that treatment can be discontinued if needed, and this approach may be used in individuals who fail to generate an adequate immune response to the antigen, a phenomenon common in the elderly who are the target population for AD therapy. The half-life of IgG in plasma is 23 days and any detrimental effects of the therapy would then presumably subside within months, whereas active immunization is irreversible and the development of an autoimmune disease is of a concern. On the other hand, although antibody administration is appropriate for certain diseases that can be treated within a relatively short period, it is less feasible for chronic use. Repeated antibody injections can lead to an antibody response, and the resulting serum immune complexes can deposit in blood vessels leading to vasculitis

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and/or glomerulonephritis. Since amyloid deposition is a process that occurs over decades, a passive immunization approach would require multiple applications, increasing the probability of these adverse effects. The use of single chain antibodies may be a safer alternative. Regardless of their relative safety/efficacy, both approaches are likely to be more effective as a preventive measure because of neuronal loss and increased amyloid burden observed in the later stages of the disease. 7. Anti-A␤ antibodies in AD Prior to and following the report of antibody-mediated reduction in plaque burden in Tg mice [36], several studies have measured the presence of anti-A␤ antibodies in AD patients. The reports have been inconsistent [5,10,14,27,42,53], and are often difficult to evaluate because of their preliminary nature. The most comprehensive of these studies reported by Hyman et al. [14] from 365 individuals showed that neither the presence nor the level of anti-A␤ antibodies correlate with the likelihood of developing dementia or with plasma levels of A␤. Because of both the low anti-A␤ titer in humans and the extensive variance in titer and A␤ levels between individuals, it will be necessary to perform repeated measures in the same individual before and after the onset of the disease to evaluate the importance of immune response to A␤ in AD. The outcome of such a study on the effect of the immune system on AD onset and/or progression will, however, only partially predict if A␤-based AD vaccination will have therapeutic value, since the titer obtained with a successful vaccination approach is much higher than in untreated individuals who have developed an autoimmune response towards A␤. The next generation of A␤ derived vaccines will likely have built-in adjuvanticity such as by using Th epitopes, carriers and lipid moieties. These approaches should enhance antibody production, particularly in the elderly who often have diminished immune response. 8. Conclusion Current data from various laboratories indicates that A␤-targeted therapy is the most likely to be effective at reducing amyloid-related pathology in AD. Clearance of soluble A␤ within the peripheral system by antibodies or A␤ chaperones may be a critical part of the pathway that reduces cerebral plaque burden in Tg mice and ultimately in AD patients. Acknowledgments Supported by NIH grants AG20197, AG20245, AG05891, the Alzheimer’s Association and Mindset Biopharmaceuticals.

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