Peptide Conjugate Compositions And Methods For The Prevention And Treatment Of Alzheimer's Disease

Abstract

The invention provides compositions and methods for the treatment of diseases associated with amyloid deposits of A.beta. in the brain of a patient, such as Alzheimer's disease. Such methods entail administering an immunogenic fragment of A.beta., lacking a T-cell epitope, capable of inducing a beneficial immune response in the form of antibodies to A.beta.. In another aspect, the immunogenic fragment of A.beta. is capable of elevating plasma A.beta. levels. The immunogenic fragments comprise linear or multivalent peptides of A.beta.. Pharmaceutical compositions comprise the immunogenic fragment chemically linked to a carrier molecule which may be administered with an adjuvant.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to composition and methods for the prevention and treatment of amyloidogenic diseases and, in particular, Alzheimer's disease.

BACKGROUND OF THE INVENTION

[0002] Alzheimer's disease (AD) is characterized by progressive memory impairment and cognitive decline. Its hallmark pathological lesions are amyloid deposits (senile plaques), neurofibrillary tangles and neuronal loss in specific brain regions. The amyloid deposits are composed of amyloid beta peptides (A.beta.) of 40 to 43 amino acid residues, which are the proteolytic products of the amyloid precursor protein (APP). Neurofibrillary tangles are the intracellular filamentous aggregates of hyperphosphorylated tau proteins (Selkoe, Science, 275: 630-631, 1997).

[0003] The pathogenesis of AD has not been fully understood, but it is expected to be a multi-factored event. Accumulation and aggregation of A.beta. in brain tissue is believed to play a pivotal role in the disease process, also know as the amyloid cascade hypothesis (Golde, Brain Pathol., 15: 84-87, 1995). According to this hypothesis, A.beta., particularly A.beta..sub.42, is prone to form various forms of aggregates, ranging from small oligomers to large, elongated profibrile structures. These aggregates are neurotoxic and are responsible for the synaptic pathology associated with the memory loss and cognition decline in the early stage of the disease (Klein et al., Neurobiol. Aging, 25: 569-580, 2004). A recent publication suggests that reduction of A.beta. in a triple transgenic mouse model also prevents intracellular tau deposition (Oddo et al., Proc. Neuron, 43:321-332, 2004). This finding suggests that the extracellular amyloid deposition may be causative for subsequent neurofibrillary tangle formation, which may in turn lead to neuronal loss.

[0004] Immunization of APP transgenic mice with A.beta. antigen can reduce the brain A.beta. deposits and mitigate disease progression. This was first reported by Shenk et al., Nature, 400: 173-177, 1999, and has now been corroborated by a large number of studies involving different transgenic animal models, various active vaccines as well as passive imrannization with A.beta. specific monoclonal antibodies (Bard et al., Nature Med, 6: 916-919, 2000; Janus et al., Nature 408: 979-982, 2000; Morgan et al., Nature, 408: 982-985, 2000; DeMattos et al., Proc. Natl. Acad. Sci., 98: 8850-8855, 2001; Bacskai et al., J. Neurosci., 22: 7873-7878, 2002; Wilcock et al., J. Neurosci, 23: 3745-3751, 2003). Consistent with these animal data, three published evaluations of postmortem human brain tissues from patients who had previously received active immunization with a pre-aggregated A.beta. (1-42) immunogen (AN1792, Betabloc) showed regional clearance of senile plaques (Nicoll et al., Nature Med., 9: 448-452, 2003; Ferrer et al., Brain Pathol., 14: 11-20, 2004; Masliah et al., Neurology, 64: 129-131, 2005). This data collectively indicates that vaccines that effectively elicit antibody responses to A.beta. antigens are efficacious against the pathological senile plaques found in AD. However, the mechanism of vaccine or antibody efficacy remains to be defined.

[0005] The most advanced immunotherapy-based AD program in the public domain had been an active immunization Phase II vaccine trial using AN1792 (Betabloc), a vaccine composed of pre-aggregated A.beta. (1-42) co-administered with the adjuvant, QS-21.TM. (Antigenics, New York, N.Y.). In January 2002, this study was terminated when four patients showed symptoms consistent with meningoencephalitis (Senior, Lancet Neurol., 1: 3, 2002). Ultimately, 18 of 298 treated patients developed signs of menigoencephalitis (Orgogozo et al., Neurology, 61: 46-54, 2003). There was no correlation between encephalitis and antibody titer and it has been reported that the likely causative mechanism for this effect was activation of T-cells to the self-immunogen, particularly the mid- and carboxy-terminal portion of the A.beta..sub.42 peptide (Monsonego et al., J. Clin. Invest., 112: 415-422, 2003). In support of this conclusion, postmortem examination of brain tissues from two vaccine recipients that developed encephalitis revealed substantial meningeal infiltration of CD4.sup.+ T cells in one patient (Nicoll et al., Nature Med., 9: 448-452, 2003) and CD4.sup.+, CD8.sup.+, CD3.sup.+, CD5.sup.+, CD7.sup.+ T cells in the other (Ferrer et al., Brain Pathol., 14: 11-20, 2004).

[0006] Current evidence suggests that increases in plasma A.beta. levels following passive or active immunization reflect the initiation of a peripheral sink as a precursor to subsequent decreases in brain A.beta.. The peripheral sink refers to a change in the equilibrium of brain and plasma A.beta. stores resulting in a net efflux of central A.beta. to the periphery (see, for example, Deane et al., J. Neurosci., 25: 11495-11503, 2005; DeMattos et al., Pro. Natl. Acad. Sci. USA, 98: 8931-8932, 2001). Other studies suggest that this increase in plasma A.beta. observed following anti-A.beta. immunotherapy is necessary for subsequent decreases in central A.beta. to be realized (Cribbs et al., 7th International Conference on AD/PD, Sorrento, Italy, 2005). Thus, when two amino acids within A.beta. are substituted (for example, such as occurs with the Dutch and Iowa mutations) the peptide is no longer able to cross from central to peripheral compartments (Davis et al., Neurobiol. Aging, in press, available on line 18 Aug. 2005). When mice expressing this mutant form of A.beta. and the Swedish mutation were immunized, no elevations in plasma A.beta. were found and no subsequent lowering of brain A.beta. was noted. By contrast, mice expressing the wild-type human A.beta. sequence plus the Swedish mutation responded to active immunization with both increases in plasma A.beta. and subsequent decreases in central A.beta. (Cribbs et al., 7th International Conference on AD/PD, Sorrento, Italy, 2005). Accordingly, it is expected that any active vaccine immunogen capable of generating an immune response that results in the elevation of plasma A.beta. levels will be useful for the treatment of Alzheimer's disease and related disorders characterized by elevated brain A.beta. levels.

[0007] Applicants herein have surprisingly found that an antigen which eliminated T-cell epitopes, to avoid a self T-cell response, is immunogenic and elevates plasma A.beta. levels. This represents a potential means to produce a safe and effective AD vaccine. Applicants herein provide such an antigen and a formulation for use as an AD vaccine.

SUMMARY OF THE INVENTION

[0008] In one embodiment, the invention provides a pharmaceutical composition comprising an immunogenic fragment of A.beta., lacking a T-cell epitope, capable of inducing an immune response in the form of antibodies to A.beta.. In one aspect, this composition comprises linear 8 amino acid peptides (8-mers) of A.beta.. In still another aspect, this composition comprises multivalent linear 8-naers interspersed with at least one spacer or a multivalent branched multiple antigenic peptide (MAP). The pharmaceutical composition can be used as a vaccine for AD and related amyloid diseases.

[0009] In another embodiment of the invention, the pharmaceutical composition is an A.beta. plasma elevating agent comprising an immunogenic fragment of A.beta., lacking a T-cell epitope, capable of inducing an immune response in the form of antibodies to A.beta. that elevate plasma A.beta. levels. The pharmaceutical composition can be used as a vaccine for AD and related amyloid diseases characterized by elevated brain A.beta. levels.

[0010] In still another embodiment of the invention, the pharmaceutical composition is linked to a carrier molecule to form a conjugate, wherein the carrier helps to elicit an immune response comprising antibodies to the A.beta. fragment. In a preferred embodiment of the invention, the carrier is the outer membrane protein complex of Neisseria meningitides (OMPC).

[0011] In a further embodiment of the invention, the pharmaceutical composition is administered with a pharmaceutically acceptable adjuvant. In a preferred embodiment the adjuvant is an aluminum adjuvant (Merck alum adjuvant, MAA) or a saponin-based adjuvant (ISCOMATRIX.RTM., CSL Ltd., Parkville, Australia).

[0012] In still another embodiment, the invention provides methods for preventing or treating a disease associated with amyloid deposits of A.beta. in the brain of a patient. Such diseases include Alzheimer's disease, Down's syndrome, cognitive impairment or other forms of senile dementia. The method comprises administering an immunogenic fragment of A.beta., lacking a T-cell epitope, selected from the group consisting of linear 8 amino acid peptides (8-mers), a multivalent linear peptides interspersed with at least one spacer and a multivalent branched multiple antigenic peptide (MAP). In a preferred embodiment the immunogenic fragment comprises a multivalent linear peptide with a polyethylene glycol (PEG) spacer. In a more preferred embodiment the immunogenic fragment comprises a multivalent branched MAP, A.beta. (3-10)/(21-28) conjugate, Construct No. 12, FIG. 6A, conjugated to OMPC.

[0013] Such methods entail the administration of an effective dose of an immunogenic fragment of A.beta., lacking a T-cell epitope, to patients in need of such treatment that will induce an immune response in the form of antibodies to A.beta.. Said antibody response is capable of elevating plasma A.beta. levels. In another aspect of this embodiment, the immunogenic fragment to be administered is linked to a carrier molecule. In yet another aspect of this embodiment, the immunogenic fragment is administered with an adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 represents synthetic 8-amino acid peptides (8-mers) (SEQ ID NOS: 2-36) derived from A.beta. (1-42) (SEQ ID NO: 1) from which peptides were selected to conduct a linear peptide scan to identify the epitopes of A.beta..

[0015] FIG. 2 represents the 8-mers selected for conjugation to KLH (FIG. 2A) and OMPC (FIG. 2B).

[0016] FIG. 3 represents the immunogenicity of selected A.beta. conjugates, described in FIG. 2, after the first (PD1), second (PD2) and third dose (PD3).

[0017] FIG. 4 represents the cross-reactivity of sera extracted from a guinea pig previously immunized with an A.beta. (3-10)-KLH conjugate (SEQ ID NO: 40) on human AD brain tissue. FIG. 4A shows immunoreactivity of the anti-A.beta. monoclonal antibody 6F3D (which recognizes amino acids 8-17 of A.beta.). The staining pattern reveals extensive amyloid pathology in this human brain. FIG. 4B demonstrates a lack of immunoreactivity of this same brain to the pre-immune sera from the immunized guinea pig prior to immunization FIG. 4C shows the immunoreactivity of the sera from an immunized guinea pig following immunization

[0018] FIG. 5 shows representative multivalent linear 8-mer peptides, which were selected based on the immunogenicity of the separate 8-mers in guinea pig studies (Example 3). These conjugates were synthesized as described and conjugated to OMPC (Example 1.J and 1.K).

[0019] FIG. 6 shows representative multivalent branched MAP conjugates, which were selected based on the immunogenicity of the separate 8-mers in guinea pig studies (Example 3). FIG. 6A shows representative divalent MAPs and FIG. 6B shows representative bromoacetyl-cysteine MAPs. These conjugates were synthesized as described and conjugated to OMPC (Example 2).

[0020] FIG. 7 represents the anti-A.beta..sub.40 titer from sera collected from rhesus monkeys following 1 (PD1) or 2 (PD2) injections with an A.beta. (1-18) peptide conjugated to OMPC formulated in Merck alum alone or Merck alum plus IMX (ISCOMATRIX.RTM., CSL, Ltd., Parkville, Australia) as an adjuvant.

[0021] FIG. 8 represents the increase in plasma A.beta. levels following administration of a A.beta. conjugate. FIG. 8A shows a greater than three-fold elevation following administration of a MAP construct comprising A.beta. (3-10)/(21-28) (Construct No. 12, FIG. 6A) conjugated to OMPC versus the monomeric constructs, A13 (3-10) (SEQ ID NO: 69) and A.beta. (21-28) (SEQ ID NO: 73) (.quadrature., Construct No. 12, FIG. 6A; , A.beta. (3-10) (SEQ ID NO 69), .tangle-solidup., A.beta. (21-28) (SEQ ID NO: 73). FIG. 8B shows that plasma A.beta. levels are independent of titer levels (.quadrature., Construct No. 12, FIG. 6A; , A.beta. (3-10) (SEQ ID NO 69), .tangle-solidup., A.beta. (21-28) (SEQ ID NO: 73).

DEFINITIONS

[0022] The term "8-mer" refers to an eight amino acid peptide which corresponds to a fragment of A.beta., an analog of a natural A.beta. peptide or a peptide mimetic. One or more 8-mers may be combined with at least one spacer to form a multivalent linear peptide or to form a multivalent branched MAP.

[0023] The term "A.beta. conjugate" means an 8-mer or immunogenic fragment of A.beta. that is chemically or biologically linked to a carrier, such as keyhole limpet hemocyanin or the outer membrane protein complex of Nesseria meningitidies (OMPC).

[0024] The term "A.beta. peptide" means any of the A.beta. peptides described herein, including, but not limited to, linear 8-mers, multivalent linear peptides with at least one spacer and multivalent branched multiple antigenic peptides (MAPS).

[0025] The term "epitope" refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. T-cell epitopes consist of peptides which are capable of forming complexes with host MHC molecules. T-cell epitopes for a human MHC class I molecules, which are responsible for induction of CD8.sup.+ T-cell responses, generally comprise 9 to 11 amino acid residues, while epitopes for human MHC class II molecules, which are responsible for CD4.sup.+ T-cell responses, typically comprise 12 or more amino acid residues (Bjorkman et al. Nature 329:506-512, 1987; Madden et al. Cell 75:693-708; Batalia and Collins; Engelhard Annu Rev Immunol., 12: 181-207-622. 1995; Madden, Annu Rev Imraunol, 13:587-622. 1995). Unlike T cells, B cells are capable of recognizing peptides as small as 4 amino acids in length. It is the T-cell epitope/MHC complexes that are recognized by T-cell receptors leading to T cell activation.

[0026] The term "immunogenic fragment of A.beta." or "immunogenic fragment of A.beta., lacking a T-cell response," as used herein refers to an 8-mer or an A.beta. fragment that is capable of inducing an immune response in the form of antibodies to A.beta., but which response does not include a T-cell response to the self antigen, A.beta..

[0027] The term "immunological" or "immune" or "immunogenic" response refers to the development of a humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against an antigen in a vertebrate individual. Such a response can be an active response induced by administration of an immunogen or a passive response induced by administration of an antibody.

[0028] The term "multivalent peptide" refers to peptides having more than one antigenic determinant.

[0029] The term "pharmaceutical composition" means a chemical or biological composition suitable for administration to a mammalian individual. As used herein, it refers to a composition comprising 8-mers, immunogenic fragments of A.beta. and A.beta. conjugates described herein to be administered optionally with or without an adjuvant.

DETAILED DESCRIPTION OF THE INVENTION

[0030] As previously described, preclinical studies suggest that active immunization resulting in an anti-A.beta. polyclonal antibody response provides efficacy against the pathological and cognitive symptoms associated with AD in transgenic mice that overexpress the amyloid precursor protein (Bard et al., Nature Med., 6: 916-919, 2000; Janus et al., Nature, 408: 979-982, 2000; Morgan et al., Nature, 408: 982-985, 2000; DeMattos et al., Proc. Natl. Acad. Sci., 98: 8850-8855, 2001; Bacskai et al., J. Neurosci., 22: 7873-7878, 2002; Wilcoc, et al., J. Neurosci., 23: 3745-3751, 2003). These preclinical studies are supported by a single clinical trial where an aggregate form of A.beta..sub.42 was used as an active immunogen. Preliminary evidence from this study suggests that pathological (Nicoll et al., Nature Med., 9: 448-452, 2003; Ferrer et al., Brain Pathol., 14: 11-20, 2004; Masliah et al., Neurology, 64:129-131, 2005) and cognitive improvements (Gilman et al., Neurology, 64 (9): 1553-1562, 2005) were found following treatment. While these findings are encouraging and consistent with preclinical studies, the treatment proved unsafe and was terminated following the appearance of meningoencephalitis in approximately 6% of the treated patients (Orgogozo et al., Neurology, 61: 46-54, 2003). Thus, there exists a need for active immunization procedures capable of an efficacious immune response and devoid of adverse safety issues.

[0031] Progress in understanding the nature of the adverse events in this preliminary clinical trial has been made. Several investigators have now reported the presence of CD4.sup.+ and CD8.sup.+ positive meningeal infiltrates on post-mortem evaluation (Nicoll, et al., Nature Med., 9: 448-452, 2003; Ferrer et al., Brain Pathol., 14: 11-20, 2004) suggestive of a T-cell response directed at the self-peptide A.beta..sub.42. However, while those skilled in the art would recognize the need to avoid a self-directed T-cell response while maintaining an appreciable antibody response (B-cell mediated), the means to produce an agent having this property is not known. This difficulty is compounded by a lack of predictive animal models or other preclinical assays with predictive validity for these activities.

[0032] To this end, Applicants herein used the differing nature of T and B cell epitopes to design the peptides used for the invention. The vaccine constructs were designed, by restricting the linear peptide size to eight amino acids and, if necessary, removing any potential C-terminal T-cell epitope anchor residues.

[0033] Accordingly, one aspect of the present invention was the identification of A.beta. fragments that are immunogenic, but lack a T-cell epitope, for use as an AD vaccine. Prior to the present application, it was not definitively known which amino acid fragments of the A.beta. peptide would produce an immunogenic response that would also be deficient in a T-cell epitope. Those skilled in the art would appreciate that previous teachings in the field did not predict, for example, that an 8-mer would produce an immunogenic response and did not distinguish the usefulness of fragments from different regions of the A.beta. peptide. See, for example, U.S. Pat. Nos. 6,808,712 and 6,787,144.

[0034] An additional aspect of the invention herein includes the identification of A.beta. plasma elevating agents comprising an immunogenic fragment of A.beta., lacking a T-cell epitope, that induce an immune response in the form of antibodies to A.beta. and that elevate plasma A.beta. levels. Such agents can be used as an AD vaccine and for related amyloid diseases characterized by elevated brain A.beta. levels. Prior to Applicants' invention, it was not known or predictable which immunogenic fragments of A.beta. would result in elevated plasma A.beta. levels. Without wishing to be bound by any theory, it is believed that the A.beta. plasma elevating agents described herein act to induce an immune response in the form of antibodies to A.beta. that, according to the peripheral sink theory of A.beta. clearance, produce elevated levels of plasma A.beta. that leads to subsequent decreases in brain A.beta.. Moreover, while individual 8-mers or immunogenic fragments of A.beta. may be capable of inducing an immune response such that plasma A.beta. levels are elevated, Applicants found that a multivalent branched MAP, A.beta. (3-10)/(21-28) (Construct No. 12, FIG. 6A), conjugated to OMPC, was particularly effective in elevating plasma A.beta. levels relative to those of its constituent monomeric constructs, A.beta. (3-10) (SEQ ID NO: 69) or A.beta. (21-28) (SEQ ID NO: 73).

Amyloid Diseases

[0035] The invention provides compositions and methods for prophylactic and therapeutic treatment of disease characterized by accumulation of amyloid deposits. Amyloid deposits comprise a peptide aggregated to an insoluble mass. The nature of the peptide varies in different disease but in most cases, the aggregate has a .beta.-pleated sheet structure and stains with Congo Red dye. Diseases characterized by amyloid deposits include Alzheimer's disease (AD), both late and early onset. In both diseases, the amyloid deposit comprises a peptide termed amyloid beta (A.beta.), which accumulates in the brain of affected individuals. Thus, the term "amyloid disease" also refers to disease characterized by elevated brain A.beta. levels.

Therapeutic Agents

[0036] Therapeutic agents for use in the present invention induce an immune response in the form of antibodies to A.beta.. Induction of an immune response can be active as when an immunogen is administered to induce antibodies or T cells reactive with A.beta. in an individual or passive, as when an antibody is administered that itself binds to A.beta. in the individual.

[0037] The therapeutic agent to be used in preventing or treating amyloid diseases, such as AD, include peptide fragments of A.beta., which can be any of the naturally occurring forms (i.e. A.beta.39, A.beta.40, A.beta.42, A.beta.42, or A.beta.43). These sequences are known in the art, see, for example, Hardy et al., TINS 20: 155-158, 1997.

[0038] As used herein, the therapeutic agent is, in a preferred embodiment, an immunogenic fragment, lacking a T-cell epitope, capable of inducing an immune response in the form of antibodies to A.beta.. The immunogenic fragment of A.beta. can be in the form of an 8-mer, a multivalent linear A.beta. conjugate having at least one PEG spacer or a multivalent branched MAP A.beta. conjugate. The therapeutic agent can be administered in the form of a pharmaceutical composition. In an another embodiment, the therapeutic agent is an A.beta. plasma elevating agent capable of inducing an immune response in the form of antibodies to A.beta. and that elevate plasma A.beta. levels in an individual. Such agents can comprise a naturally occurring peptide fragment or may include one or more substitutions, additions or deletions, and may include synthetic or non-naturally occurring amino acids. Fragments and constructs can be screened for prophylactic and therapeutic efficacy in the assays described in the examples herein.

[0039] While in a preferred embodiment the therapeutic agents comprise a peptide fragment of A.beta., such agents may also include peptides and other compounds that do not necessarily have a significant amino acid sequence similarity with A.beta., but that nevertheless can serve as mimetics of A.beta. and induce a similar immune response. For example, peptides and proteins forming .beta.-pleated sheets can be screened for suitability for the invention herein. Similarly, combinatorial libraries and other compounds can be screened for suitability for the invention herein.

[0040] Such identified therapeutic agents can be linked either chemically or biologically to a carrier to facilitate their use as an immunogen. Such carriers include serum albumins, keyhole limpet hemocyanin (KLH), immunoglobulin molecules, ovalbumin, tetanus toxoid protein, or a toxoid from other pathogenic bacteria, such as diphtheria, E. coli, cholera, or H. pylori, or an attenuated toxin derivative. In a preferred embodiment of the invention the carrier is the outer membrane protein complex of Neisseria meningitides (OMPC).

[0041] The invention herein also contemplates the use of such therapeutic agents in a pharmaceutical composition comprising an 8-mer or immunogenic fragment of A.beta., which may be linked to a carrier, to be administered optionally with an adjuvant. Suitable adjuvants include aluminum salts (alum), a lipid, such as 3 De-O-acylated monophosphoryl lipid A (MPL) or a saponin-based adjuvant. In a preferred embodiment the adjuvant is an aluminum adjuvant (Merck alum adjuvant, MAA) or a saponin-based adjuvant (ISCOMATRIX.RTM., CSL Ltd, Parkville, Australia.

Treatment Regimes

[0042] Effective doses of the compositions of the invention herein for the prophylactic or therapeutic treatment of AD and other amyloid diseases will vary depending upon many factors including, but not limited to, means of administration, target site, physiological state of the patient, other medications administered and whether treatment is a therapeutic, i.e. after on-set of disease symptoms, or prophylactic, i.e. to prevent the on-set of disease symptoms. In a preferred embodiment the patient is human and the therapeutic agent is to be administered by injection.

[0043] The amount of immunogen or therapeutic agent to be employed will also depend on whether an adjuvant is to be administered either concomitantly or sequentially, with higher doses being employed in the absence of an adjuvant.

[0044] The amount of an immunogen or therapeutic agent to be administered will vary, but amounts ranging from 0.5-50 .mu.g of peptide (based on the A.beta. peptide content) per injection are considered for human use. Those skilled in the art would know how to formulate compositions comprising antigens of the type described herein.

[0045] The administration regimen would consist of a primary immunization followed by booster injections at set intervals. The intervals between the primary immunization and the booster immunization, the intervals between the booster injections, and the number of booster immunizations will depend on the antibody titers and duration elicited by the vaccine. It will also depend on the functional efficacy of the antibody responses, namely, levels of antibody titers required to prevent AD development or exerting therapeutic effects in AD patients. A typical regimen will consist of an initial set of injections at 1, 2 and 6 months. Another regimen will consist of initial injections at 1 and 2 months. For either regimen, booster injections will be given either every six months or yearly, depending on the antibody titers and durations. An administration regimen can also be on an as-needed basis as determined by the monitoring of immune responses in the patient.

Identification of AD Vaccine Epitopes.

[0046] In order to determine which 8-amino acid fragments ("8-mers") of the A.beta. peptide were sufficient to produce an immunogenic response, Applicants systematically scanned the entire length of A.beta..sub.42 with small (8 amino acids) overlapping synthetic peptides derived from the naturally occurring A.beta. sequence (SEQ ID NO. 1) as shown in FIG. 1 (SEQ ID NOS: 2-37). Twenty nine overlapping eight amino acid peptides, spanning the entire length of A.beta..sub.42, were synthesized (FIG. 2A) for use as antigens. To improve solubility, several of the peptides were modified by the addition of triple lysine (KKK) (SEQ ID NOS: 52, 53, 54, 56, 59, 60, 62, 64 and 65) or glutamine (EEE) (SEQ ID NOS: 50, 51 and 61) residues or the use of a polyethelyene glycol (PEG) (SEQ ID NOS: 55 and 63) spacer: For this reason, peptides spanning the sequences of A.beta. corresponding to residues (11-18) and (13-20) were made in multiple forms, the first with a 6-aminohexanoic acid (Aha) spacer plus a functional group for chemical cross-linking at N-terminus and the other form with Aha and the functional group at C-terminus. As a control, Applicants included a longer peptide, A.beta. (1-18).

[0047] As used herein, the immunogenic fragments may be 8-mer peptides (eight amino acid residues) derived from the naturally occurring, i.e. wild type, or synthetic A.beta. (SEQ ID NO:1) or any mutation or variation thereof. Such mutation or variant can be produced by synthetic or recombinant means known to those of ordinary skill in the art. One example of such a variant is the EV substrate (EVEFRHDSGYEVHFIQKLVFFAEDVGSNKGAIIGLMVGGVVIA) (SEQ ID NO: 66) a peptide corresponding to A.beta. (1-42) in which positions 1 and 2 of wild type A.beta. have been varied. A.beta. conjugates for use in vaccine formulation

[0048] Selection of A.beta. conjugates for use in formulating a vaccine was based on the immunogenicity of the 8-mers. In order to determine the immunogenicity of the 8-mer in a species with a sequence identical to the human A.beta. sequence, the 29 peptides (FIG. 2A) were conjugated to KLH to form an A.beta. conjugate and tested in guinea pigs (FIG. 3). As a control immunogen, A.beta. (1-18)-KLH (SEQ ID NO: 37) was included in this analysis.

[0049] Guinea pigs were immunized as described in Example 3.B with conjugated immunogens formulated in alum plus 50 .mu.g of ISCOMATRIX.RTM. (CSL, Ltd., Parkville, Australia). In order to distinguish immunogenic from non-immunogenic A.beta..sub.42 fragments, guinea pigs were immunized three times at four week intervals. Three weeks after each immunization, blood samples were collected and tested by ELISA for antibody titers against A.beta..sub.40 peptide. These titers are shown in FIG. 3 as post-dose 1 (PD1), post-dose 2 (PD2) and post-dose 3 (PD3), respectively.

[0050] Following the first injection (PD1) some peptide regions elicited appreciable antibody titers as did the 18-mer control. In particular, A.beta. conjugates corresponding to A.beta. amino acids 1-8,2-9, 3-10, 17-24, 21-28, and 33-40 all produced titers in excess of 1:800. After the second injection (PD2), 15 of the A.beta. conjugates elicited antibody titers in excess of 1:1000. Analysis at post-dose 3 (PD3) further confirmed that certain regions of A.beta. are more immunogenic relative to others. Eleven regions demonstrated titers greater than 1:6000. These included regions corresponding to A.beta. amino acids 1-8, 3-10, 7-14, 11-18, 13-20, 15-22, 19-26, 21-28, 23-30, 27-34 and 29-36. Of these regions, five regions were highly immunogenic (>1:10000) including: regions 1-8, 15-22, 21-28, 23-30 and 29-36. This data suggests that certain 8-amino acid regions of A.beta. are highly immunogenic, while other regions (e.g., 5-12, 25-32, 31-38 and 35-42) are non-immunogenic (titers <1:300). The results also demonstrate that while the A.beta. conjugates were capable of eliciting an A.beta..sub.40 peptide-specific antibody response, not all fragments of A.beta. were equally immunogenic.

Immunoreactivity of A.beta. Peptide-KLH Conjugates

[0051] In order to demonstrate that the immune sera generated from the guinea pigs following immunization with the A.beta. peptide-KLH conjugates is relevant to human AD, a study was performed to evaluate the immunoreactivity of polyclonal sera from a guinea pig immunized with an A.beta. (3-10)-KLH (SEQ ID NO: 40) conjugate. The serum sample collected four weeks following the second injection of A.beta.0 (3-10)-KLH (SEQ ID NO: 40) conjugate from a guinea pig was tested for reactivity with human AD brain tissues by immunohistochemistry (Example 4).

[0052] As depicted in FIG. 4 the immunogenic response produced by the A.beta. (3-10)-KLH (SEQ ID NO: 40) conjugate produced an antibody response that was directed against human AD brain tissue. FIG. 4A demonstrates immunoreactivity of the monoclonal anti-A.beta. antibody 6F3D (Vector Laboratories). As shown, this brain has extensive A.beta. deposits in a manner expected to be typical for human AD. FIG. 4B demonstrates a lack of immunoreactivity of sera from a pre-immunized guinea pig. FIG. 4C shows positive immunoreactivity of sera from this same guinea pig following two injections of the A.beta. (3-10)-KLH (SEQ ID NO: 40) conjugate. Collectively, this data demonstrates that the immunogenicity found by ELISA contains a significant antibody response directed against human A.beta. found in this AD tissue. These results confirm and extend the unexpected finding of the differential immunogenicity imparted by particular fragments of A.beta. to further demonstrate that this response is directed in a manner consistent with a therapeutic application.

Generation of OMPC Conjugates and Multiple Antigenic Conjugates

[0053] On the basis of immunogenicity in guinea pigs, the relative location of the peptide fragment within the A.beta..sub.42 amino acid sequence, the solubility of the A.beta. fragments and the feasibility of using OMPC as a carrier protein, Applicants selected seven 8-mers (FIG. 2B) for OMPC conjugation. These peptide fragments correspond to the following amino acid regions of A.beta.: 1-8, 2-9, 3-10, 7-14, 17-24, 21-28 and 33-40.

[0054] The invention described herein includes multivalent peptide conjugates such as those shown in FIGS. 5, 6A and 613. Multivalent branched MAP-OMPC conjugates (FIGS. 6A and 6B) were generated by using a lysine-based scaffold, whereas multivalent linear 8-mer-OMPC conjugates (FIG. 5) were prepared using a PEG linker. Those skilled in the art will appreciate that a PEG linker, compared to conventional amino acid linkers that can also be used herein, offers the advantage of lower immunogenicity and greater peptide solubility. In a preferred embodiment of the invention, the immunogenic fragment is a multivalent MAP conjugated to OMPC. It should be understood by those skilled in the art that such a conjugation is not a 1:1 ratio of peptide to carrier. Rather, a plurality of peptides is attached in a spherical manner to each OMPC molecule. It will be further appreciated by those skilled in the art that the use of multivalent linear constructs and MAPs will enhance solubility, formulation stability, immunogenicity and the diversity of the polyclonal response.

Immunogenicity of OMPC Conjugate Vaccines

[0055] In an effort to evaluate the immunogenicity of an A.beta. peptide-OMPC conjugate and to further evaluate the benefit of an adjuvant with this vaccine construct, Applicants initiated a study in rhesus monkeys. Rhesus monkeys were vaccinated with an A.beta. (1-18)-OMPC conjugate (dose based on the A.beta. peptide content), which was formulated either in Merck alum adjuvant (MAA) or MAA and ISCOMATRIX.RTM. (CSL, Ltd., Parkville, Australia). Blood samples were collected and used to determine the antibody titers against A.beta..sub.40. Interim analysis of this ongoing study demonstrated that at post-dose 1 (PD1) the monkeys receiving 5 .mu.g vaccine in alum failed to develop any detectable titers, while those receiving 30 .mu.g vaccine in alum developed low A.beta..sub.40 specific titers. All monkeys that received the alum plus ISCOMATRIX.RTM. formulation developed significant antibody titers. At post-dose 2 (PD2) both doses of the A.beta. conjugate in alum alone produced similar titer levels, whereas the cohorts receiving the alum plus ISCOMATRIX.RTM. developed 10-fold higher antibody titers relative to the alum alone cohorts. The results of this study confirmed that the A.beta.-OMPC conjugate is immunogenic in non-human primates. The data further demonstrated that the efficacy of such a conjugate vaccine is significantly enhanced by a saponin-based adjuvant such as ISCOMATRIX.RTM..

EXAMPLES

Example 1

Preparation of A.beta. Conjugates

[0056] This example describes the preparation of A.beta. peptide fragments subsequently used for the A.beta. conjugates to induce an immune response in the form of antibodies to A.beta..

A. Preparation of A.beta. (8-mers) Peptides (SEQ ID NOS.: 37-65; FIG. 2A)

[0057] The peptides intended for conjugation to maleimide derivatized carrier proteins were synthesized with a cysteine residue at the carboxy terminus. The spacer, Aha (6-aminohexanoic acid) was incorporated between the primary peptide sequence and the carboxy terminal cysteine as a structural element for minimizing steric accessibility to carrier protein during conjugation. Additionally, solubilizing residues represented by EFF, KKK or PEG were introduced at the C-terminus in sequences 14,15,16 17,18,19,20,23,24,25,26,27,28,29. The PEG unit was introduced as, O--(N--Fmoc-2-aminoethyl)-O'-(2-carboxyethyl)-undecaethyleneglycol [Fmoc-NHCH.sub.2CH.sub.2--O--(CH.sub.2CH.sub.2O).sub.10CH.sub.2CH.sub.2OC- H.sub.2CH.sub.2CO.sub.2H].

[0058] Starting with Rink Amide MBNA resin the A.beta. peptides were prepared by solid-phase synthesis on an automated peptide synthesizer using Fmoc chemistry protocols as supplied by the manufacturer (Applied Biosystems, Foster City, Calif.). Following assembly the resin bound peptide was deprotected and cleaved from the resin using a cocktail of 94.5% trifluoroacetic acid, 2.5% 1,2-ethanedithiol, 1% triisopropylsilane and 2.5% H.sub.2O. Following a two hour treatment the reaction was filtered, concentrated and the resulting oil triturated with ethyl ether. The solid product was filtered, dissolved in 50% acetic acid/H.sub.2O and freeze-dried. Purification of the semi-pure product was achieved by RPHPLC using a 0.1% TFAJH.sub.2O/acetonitrile gradient on a C-18 support. Fractions were evaluated by analytical HPLC. Pure fractions (>98%) were pooled and freeze-dried. Identity was confirmed by amino acid analysis and mass spectral analysis.

B. Preparation of A.beta. Peptide-KLH Conjugates (SEQ ID NOS.: 37-65; FIG. 2A)

[0059] For preparing the KLH conjugates, the A.beta. peptides (8-mers), 2 mg, containing a C-terminal cysteine was suspended in 1 ml of commercial maleimide conjugation buffer (83 mM sodium phosphate, 0.1 M EDTA, 0.9 M NaCl, 0.02% sodium azide, pH 7.2 (Pierce Biotechnology, Rockford, Ill.). A 2 mg sample of commercial maleimide-activated KLH (Pierce Biotechnology, Rockford, Ill.) was added to the peptide and allowed to react at 25.degree. C. for four hours. The conjugate was separated from unreacted peptide and reagents by exhaustive dialysis versus PBS buffer using 100,000 Da dialysis tubing. The amount of peptide incorporated into the conjugate was estimated by amino acid analysis following a 70 hour acid hydrolysis. Peptide concentrations were determined to be between 0.24 and 0.03 mg/ml.

C. Synthesis of Bromoacetylated A.beta. Peptides (SEQ ID NOS.: 67-77; FIG. 2B)

[0060] Bromoacetylated peptides were prepared by standard t-Boc solid-phase synthesis, using a double coupling protocol for the introduction of amino acids on the Applied Biosystems model 430A automated synthesizer. Starting with p-methylbenzhydrylanaine resin the carboxy terminal amino acid t-Boc-Lys (Fmoc)-OH was introduced followed by the subsequent amino acids in the sequence. Aha was introduced as a spacer to all of these sequences and a PEG unit in sequences 35 and 37 to aid in aqueous solubility. The PEG unit was introduced as O--(N-Boc-2-aminoethyl)-0'-(N-diglycolyl-2-aminoethyl) hexaethyleneglycol [Boc-NHCH.sub.2CH.sub.2--O--(CH.sub.2CH.sub.2O).sub.6CH.sub.2CH.sub.2NHCO- CH.sub.2OCH.sub.2CO.sub.2H]. The amino terminous was capped by the coupling of acetic acid. After assembly of the primary sequence the Fmoc protecting group on the epsilon amino group of the carboxy terminal lysine was removed by treatment with piperidine. Subsequently the N.sup..epsilon. amino group was reacted with Bromoacetic anhydride in methylene chloride as the solvent for 30 minutes. Deprotection and removal of the peptide from the resin support were achieved by treatment with liquid hydrofluoric acid and 10% anisole as a scavenger. The peptides were purified by preparative HPLC on reverse phase C-18 silica columns using a 0.1% TFA/acetonitrile gradient. Identity and homogeneity of the peptides were confirmed by analytical HPLC and mass spectral analysis.

D. Synthesis of Bromoacetylated Divalent MAP, Construct No. 8, FIG. 6A

[0061] The synthesis of bromoacetylated branched multiple antigenic peptides (MAPs) is similar to that described in Example 1.C. Following coupling of the carboxyterminal Fmoc-Lys(ivDde)-OH [ivDde=1, (4,4-Dimethyl-2,6-dioxo-cyclohexylidene)-3-methyl-butyl] to MBHA resin the .alpha.-amino Fmoc protecting group was removed using piperidine and the synthesis continued with the introduction of t-Boc-Lys(Fmoc)-OH. After deprotection of the t-Boc group the sequence was extended with the following t-Boc protected amino acids: Aha, Y, G, S, D, H, R, F, E and the amino terminous capped by coupling acetic acid on the ABI synthesizer. The side chain lysine Fmoc protecting group was removed with piperidine and the N.sup..epsilon. arm of lysine extended on the ABI synthesizer with the introduction of the following protected amino acids: Aha, H, H, V, E, Y, G, S, D and the amino terminous capped by coupling acetic acid. Removal of the ivDde protecting group was by treatment with 5% hydrazine in dimethylformamide for 5 minutes providing the unblocked N.sup..epsilon. amino group on the carboxy terminal lysine which was further elaborated with bromoacetic anhydride as described in Example 1.C. Cleavage of the peptide from the resin, its subsequent purification and characterization are as described in Example 1.C.

E. Synthesis of Bromoacetylated MAPS, Construct Nos. 11 and 12, FIG. 6A

[0062] MAP Constructs Nos. 11 and 12 were prepared as described in Example 1.D.

F. Synthesis of Cysteine Multivalent MAP, Construct No. 9, FIG. 6A

[0063] Starting with MBHA resin the following t-Boc protected amino acids were assembled on the ABI automated synthesizer C, Lys(Fmoc), Alta, Y, G, S, D, H, R, F, E followed by coupling with acetic acid. The N.sup..epsilon. amino Fmoc protecting group of lysine was removed and the synthesis continued with the introduction of the following t-Boc protected amino acids: Aha, H, H, V, E, Y, G, S, D followed by coupling with acetic acid. The resin bound peptide was isolated, purified and characterized as in Example 1.C. Note: Instead of 10% anisole as in Example 1.C, a 1:1 mixture of p-cresol: p-thiocresol was used as a scavenger during BF cleavage.

G. Synthesis of Cysteine Divalent MAPs, Construct Nos. 10, 13 and 14, FIG. 6A

[0064] Divalent MAPs, Construct Nos. 10, 13 and 14, FIG. 6A, were prepared as described in Example 6.F. The PEG unit was introduced as O--(N-Boc-2-aminoethyl)-O'--(N-diglycolyl-2-aminoethyl) hexaethyleneglycol (t-Boc-NHCH.sub.2CH.sub.2--O--(CH.sub.2CH.sub.2O).sub.6 CH.sub.2CH.sub.2NHCOCH.sub.2OCH.sub.2CO.sub.2H).

H. Synthesis of Bromoacetylated Multivalent MAP, Construct No. 16, FIG. 6B

[0065] Using the ABI automated synthesizer Fmoc-Lys (t-Boc)-OH was coupled to MBHA resin. Following removal of the t-Boc protecting group on the N.sup..epsilon. amino group of lysine the sequence was extended with the introduction of the following t-Boc protected amino acids: Aha, Y, G, S, D, H, R, F, E, followed by coupling of acetic acid. The N.alpha. Fmoc protecting group on lysine was removed by manual treatment with piperidine. The sequence was further elaborated (on ABI synthesizer) with the introduction of Fmoc-Lys (t-Boc)-OH followed by the following t-,Boc protected amino acids: Aha, H, H, V, E, Y, G, S, D and coupling of acetic acid. The lysine Fmoc N.sup..alpha. amino protecting group was removed as previously described and the synthesis continued with the introduction of Fmoc-Lys(t-Boc)-OH followed by the t-Boc protected amino acids: Aha, K, N, S, G, V, D, E, A and acetic acid coupling. The N.sup..alpha. Fmoc protecting on lysine was removed and the synthesis continued with the introduction Fmoc-Lys(t-Boc)-OH followed by the following t-Boc protected amino acids: Aha, V, V, G, G, V, M, L, G and acetic acid coupling. Following removal of the N.alpha. Fmoc protecting group of lysine the resin bound peptide was reacted with bromoacetic anhydride as in Example 1.C. Isolation and characterization of the final product was as in Example 1.C.

I. Synthesis of Multivalent MAPs, Construct Nos. 15 and 17, FIG. 6B

[0066] The synthesis of MAP A.beta. conjugates, Construct Nos. 15 and 17, FIG. 6B, are as described in Example 1.F and 1.H.

J. Synthesis of Bromoacetylated Multivalent Linear Peptide, Construct No. 1, FIG. 5

[0067] Starting with MBHA resin the primary sequence was synthesized using t-Boc chemistry on the ABI automated synthesizer as described in Example 6.A. The interspaced PEG units were manually introduced as the Fmoc-1-amino-4,7,10-trioxa 13-tridecanamine succinic acid [Fmoc-NHCH.sub.2CH,CH.sub.2--O--(CH.sub.2 CH.sub.2O).sub.2 CH.sub.2CH.sub.2CH.sub.2 NHCOCH.sub.2CH.sub.2CO.sub.2H] using BOP reagent as the coupling agent. Piperidine was used for deprotection of the Fmoc group. Brornoacetylation of the amino terminus was as described in Example 1.C. Isolation and characterization of the desired product was as in Example 1.C.

K. Synthesis of Multivalent Linear A.beta. peptides, Construct Nos. 2, 5, 6 and 7, FIG. 5

[0068] The synthesis of multivalent linear A.beta. peptides, Construct Nos. 2, 5, 6 and 7 are as described in Example 1.J.

Example 2

Chemical Conjugation of A.beta. Peptides to OMPC

[0069] This example presents the chemical conjugation of peptides derived from human A.beta..sub.42 to purified Outer Membrane Protein Complex (OMPC) of Neisseria meningitidis, type B. The chemical nature of the coupling is reaction between haloacetyl-derivatized peptide and thiol-derivatized protein of the membrane complex. Amyloid peptides were synthesized as described above with a bromoacetyl functionality on the N-terminus for divalent linear epitope peptides or on the C-terminus or attached through the epsilon amino group of a lysine residue for monovalent linear and branched MAP forms. The BrAc group was separated from the mature peptide by a spacer consisting of 6-aminohexanoic acid (Aha). Refer to sequences described above. Conjugation will be described for the representative peptide, A.beta. (3-10). All manipulation of OMPC-containing solutions was performed in a laminar flow environment following standard aseptic techniques.

A. Thiolation of OMPC

[0070] Purified, sterile OMPC, obtainable from a process such as that described in Fu, U.S. Pat. No. 5,494,808 used for the production of PedvaxHIB.RTM. and pneumococcal conjugate vaccines, was thiolated on a portion of its surface-accessible lysine residues using the reagent N-acetylhomocysteinethiolactone (NAHT, Aldrich, St. Louis, Mo.). OMPC in water, 117 mg, was pelleted by centrifugation at 289,000.times.g for 60 minutes at 4.degree. C. and the supernatant was discarded. N2-sparged activation buffer (0.11 M sodium borate, pH 11) was added to the centrifuge tube and the pellet was dislodged with a glass stir rod. The suspension was transferred to a glass Dounce homogenizer and resuspended with 30 strokes. The centrifuge tube was washed and the wash dounced with 30 strokes. Re-suspended pellet and wash were combined in a clean vessel to give a OMPC concentration of 10 mg/mL. Solid DTT and EDTA were dissolved in N2-sparged activation buffer and charged to the reaction vessel at a ratio of 0.106 mg DTT/mg OMPC and 0.57 mg EDTA/mg OMPC. After gentle mixing, NAHT was dissolved in N2-sparged water and charged to the reaction at the ratio of 0.89 mg NAHT/mg OMPC. Reaction proceeded for three hours at ambient temperature, protected from light in a N2 hood. At completion, OMPC was pelleted as described above and re-suspended at 6 mg/mL by Dounce homogenization in N2-sparged conjugation buffer (25 mM sodium borate, pH 8.5, 0.15 M NaCl) to wash the pellet. For final re-suspension, the OMPC was pelleted as above and re-suspended at 10 mg/mL by Dounce homogenization in N2-sparged conjugation buffer. An aliquot was removed for free thiol determination by Ellman assay and the bulk product was stored on ice in dark until use. Measured thiol content was between 0.2 to 0.3 .mu.mol/mL.

B. Conjugation of A.beta. Peptide to OMPC

[0071] Functional BrAc content of peptide was assumed to be 1:1 on a molar basis. Sufficient peptide was weighed to give a 1.6 molar excess of BrAc over total thiol. The targeted total OMPC protein for each conjugation was 15 mg. Peptides were re-suspended in N2-sparged conjugation buffer at 2.6 mg/mL and slowly added to thiolated OMPC solution. The reactions were protected from light and incubated at ambient temperature for about 22 hours. Residual free OMPC thiol groups were quenched with a 5-fold molar excess of N-ethylmaleimide for 18 hours at ambient temperature. A thiolated OMPC-only control was carried through the conjugation protocol in parallel. Upon completion of quenching, conjugate and control were transferred to 100,000 Da molecular weight cut-off dialysis units and dialyzed exhaustively against at least five changes of conjugation buffer. Upon completion of dialysis, samples were transferred to 15 ml polypropylene centrifuge tubes and centrifuged at 2,280.times.g for five minutes at 4.degree. C. to remove any aggregated material. Aliquots were removed for analysis and the bulk was stored at 4.degree. C.

C. Analysis of A.beta. Peptide-OMPC Conjugates

[0072] Total protein was determined by the modified Lowry assay and samples of conjugate and control were analyzed by quantitative amino acid analysis (AAA). Peptide to OMPC molar ratios were determined from quantitation of the unique residue S-carboxymethylhomocysteine which was released upon acid hydrolysis of the nascent peptide-OMPC bond. The OMPC-specific concentration was determined from hydrolysis-stable residues which were absent from the peptide sequence and thus unique to OMPC protein. Assuming 1 mol of peptide for every mol SCMHC, the ratio of SCMHC/OMPC was thus equivalent to the peptide/OMPC content. The mass loading of peptide could be calculated from this ratio using the peptide molecular weight and an average OMPC mass of 40,000,000 Da.

[0073] The covalent nature of the conjugation was qualitatively confirmed by SDS-PAGE analysis using 4-20% Tris-glycine gels (Invitrogen, Carlsbad, Calif.) where an upward shift in mobility was observed for the Coomassie-stained conjugate bands relative to control.

[0074] The calculated molar loading ratios (mol peptide/mol OMPC) for all conjugated BrAc peptides were:

TABLE-US-00001 Peptide/OMPC Peptide Peptide Mw (mol/mol) A.beta. (3-10) - BrAc 1,412 2,793 Ab (7-14) - BrAc 1,344 2,283 Ab (21-28) - BrAc 1,222 2,126 Ab (17-24) - BrAc 1,809 1,795 Ab (33-40) - BrAc 1,601 2,139 A-D-MAP-BrAc 2,498 2,173 A-B-MAP-BrAc 2,622 2,147 BrAc-linear-D-A 2,649 2,263 BrAc-linear-B-A 2,773 2,178 Ab (1-8) - BrAc 1,378 2,759 F-D-MAP-BrAc 2,463 1,318 BrAc-linear-D-F 2,615 1,812 F-G-A-D-MAP-BrAc 5,111 636

Example 3

Immunogenicity of A.beta. Conjugates

[0075] This example describes the formulation and administration of the A.beta. conjugates capable of inducing an immune response in the form of antibodies to A.beta..

A. Formulation of Vaccine Conjugates

[0076] The A.beta. peptide-KLH conjugate vaccines were formulated in ISCOMATRIX.RTM. (CSL Ltd., Parkville, Australia). All A.beta. peptide-OMPC conjugate vaccines were formulated in alum, either with or without a second adjuvant, such as the saponin-based adjuvant, ISCOMATRIX.RTM. (CSL Ltd., Parkville, Australia). All the sample manipulations were performed under sterile conditions.

[0077] For the alum formulations, conjugates are diluted one times saline at a designated peptide concentration and mixed with two times alum (Merck, Product No. 39943), which corresponds to 900 .mu.g/mL Merck alum prepared in sterile saline (150 mM sterile sodium chloride solution). Thus, target concentration in the vaccine is 450 .mu.g/mL Merck alum or one time Merck alum. Target peptide (antigen) concentrations for animal studies were as follows: for mice--12.1 .mu.g/mL, (Dose 0.1 mL); for monkeys--10 .mu.g/mL or 60 .mu.g/mL (Dose 0.5 mL) and for guinea pigs--12.5 .mu.g/mL (Dose 0.4 mL). The mix is incubated for two hours at room temperature. To obtain the injection dose, the alum-absorbed conjugates are diluted with one time alum to reach the target peptide concentration. Where a second adjuvant is needed, i.e. ISCOMATRIX.RTM., the target concentration was 10 .mu.g/ML for mice studies, 0, 100 or 200 .mu.g/mL for monkey studies and 125 .mu.g/mL for guinea pigs.

[0078] 1. ISCOMATRIX.RTM. Preparation

[0079] Using a cassette membrane (Slide-A-Lyzer Dialysis Cassett, 10K MWCO, Pierce, Rockford, Ill.), ISCOMATRIX.RTM. is dialyzed into sterile spline solution at 2-8.degree. C. Sterile saline solution is changed 2-3 times during dialysis. After completion of dialysis, ISCOMATRIX.RTM. is filter sterilized using a syringe filter (0.22 .mu.M Millex-GV syringe filter, Millipore, Billerica, Mass.). The concentration of sterile, dialyzed ISCOMATRIX.RTM. is determined by RP-HPLC. ISCOMATRIX.RTM. is stored sterile at 2-8.degree. C. until use.

[0080] 2. A.beta. peptide-OMPC Conjugate and Merck Alum Preparation

[0081] A.beta. peptide-OMPC conjugate stocks are diluted into sterile 1.times. saline solution. The diluted AD peptide-OMPC conjugate stocks are then added to 2.times. Merck alum in 1.times. sterile saline solution and mixed for one hour on a rotating wheel at room temperature. The mixture is allowed to settle on the bench top for 15 minutes at room temperature and is then centrifuged at 1500 rpm for ten minutes. The supernatant is decanted off gently (UV analysis of supernatant is performed to determine % A.beta. peptide-OMPC conjugate bound to alum) and the pellet is resuspended in sterile 1.times. saline. The mixture is aliquoted into sterile 3 mL tubing glass vials and then stored at 2-8.degree. C. until final formulation with ISCOMATRIX.RTM..

[0082] 3. Formulation of A.beta. Peptide-OMPC/Alum and ISCOMATRIX.RTM. Vaccine

[0083] Prior to final formulation with ISCOMATRIX.RTM., the particle size of the A.beta. peptide-OMPC/alum in saline is determined by static light scattering to confirm binding and monitor particle stability. The sterile, dialyzed ISCOMATRIX.RTM. in 1.times. saline is added to A.beta. peptide-OMPC/alum in sterile 150 mM NaCl while vortexing. Vials are stoppered, capped and crimped to completely seal. Vaccine is stored at 2-8.degree. C. prior to injection. Prior to injection, each vaccine is vortexed for 3-5 minutes.

[0084] B. Immunogenicity of Conjugate Vaccines in Guinea Pigs

[0085] Six to ten week old female guinea pigs were obtained from Harlan Inc., Indianapolis, Iowa and maintained in the animal facilities of Merck research Laboratories in accordance with institutional guidelines. All animal experiments were approved by Merck Research Laboratories Institutional Animal Care and Use Committee (IACUC). Antigens were prepared in phosphate-buffered saline and formulated in the designated adjuvant.

[0086] Two animals per group were immunized with the A.beta. peptide-KLH conjugates shown in FIG. 2A intramuscularly with 400 .mu.l of a conjugate vaccine (8 .mu.g by peptide content or 50 .mu.g by total conjugate) in the presence of 40 .mu.g of ISCOMATRIX.RTM.. The immunizations were performed three times in four-week intervals. Serum samples were collected before first immunization (pre-bleeds) and three weeks after each immunization and stored at 4.degree. C. prior to antibody titer determinations. The antibody titers were determined by ELISA according to the protocol that follows using A.beta..sub.40 as the target antigen.

[0087] The ELISA based analysis is as follows: Ninety six-well plates were coated with 50 .mu.l per well of A.beta. at a concentration of 4 .mu.g/ml in 50 mM bicarbonate buffer, pH 9.6, at 4.degree. C. overnight. Plates were washed with phosphate buffered saline (PBS) and blocked with 3% skim milk in PBS containing 0.05% Tween-20 (milk PBST). Testing samples were diluted in a 4-fold series in PBST. One hundred .mu.l of a diluted sample was added to each well, and the plates were incubated at 24.degree. C. for two hours and then washed six times with PBST. Fifty .mu.l of HRP-conjugated secondary antibodies at 1:5000 dilution in milk PBST was added per well and the plates were incubated at 24.degree. C. for one hour. The plates were washed three times and 100 .mu.l of 1 mg/ml o-phenylenediamine dihydrochloride in 100 mM sodium citrate, pH 4.5 was added per well. After 30 minutes incubation at 24.degree. C., the reaction was stopped by adding 100 .mu.l of 1N H.sub.2SO.sub.4 per well, and the plates were read at 490 nm using an ELISA plate reader. The antibody titer was defined as the reciprocal of the highest dilution that gave an OD490 nm value above the mean plus two standard deviations of the conjugate control wells.

[0088] The results of this analysis, shown in FIG. 3, demonstrated that following the first injection (PD1) some peptide regions elicited appreciable antibody titers as did the 18-mer control. In particular, A.beta. peptide fragments corresponding to A.beta. amino acids 1-8, 2-9, 3-10, 17-24, 21-28, and 33-40 all produced titers in excess of 1:800. After the second injection (PD2), 15 of the 8-mer conjugates elicited antibody titers in excess of 1:1000. Analysis at post-dose 3 (PD3) further confirmed that certain regions of the A.beta. peptide were more immunogenic relative to others. Eleven regions demonstrated titers greater than 1.6000. These included regions corresponding to A.beta. amino acids 1-8, 3-10, 7-14, 11-18, 13-20, 15-22, 19-26, 21-28, 23-30, 27-34 and 29-36. Of these regions, five regions were highly immunogenic (>1:10000) including: regions 1-8, 15-22, 21-28, 23-30 and 29-36. The results demonstrate that 8-mer conjugates are capable of eliciting an A.beta..sub.40 specific antibody response. Unexpectedly, and contrary to previous teachings, not all fragments of A.beta. were equally immunogenic. In fact, these data suggest that certain 8-mers are highly immunogenic, while other regions of A.beta. (e.g., 5-12, 25-32, 31-38 and 35-42) are non-immunogenic (titers <1:300).

C. Immunogenicity of Conjugate Vaccines in Rhesus Monkeys

[0089] A study was conducted in non-human primates, i.e. rhesus monkeys, comparable to that done with guinea pigs to determine whether A.beta. peptide-OMPC conjugates and an alum and ISCOMATRIX.RTM. adjuvant provided an immune response. Rhesus monkeys (Macaca mulatta) were maintained in accordance with the institutional animal care protocols of Merck Research Laboratories and New Iberia Research Center (The University of Louisiana at Lafayette, New Iberia, La.).

[0090] Applicants used A.beta. peptides conjugated to OMPC as the model antigens, including, the 8-mers shown in FIG. 2B: A.beta. (1-8) (SEQ. ID NO: 67), A.beta. (3-10) (SEQ. ID NO: 69), A.beta. (7-14) (SEQ ID NO: 70), A.beta. (17-24) (SEQ ID NO. 72), A.beta. (21-28) (SEQ ID NO: 73) and A.beta. (33-40) (SEQ ID NO. 74); the divalent linear peptides shown in FIG. 5: A.beta. (3-10) (7-14) (Construct No. 1), A.beta. (3-10) (21-28) (Construct No. 2), A.beta. (1-8)(21-28) (Construct No. 5); and the multivalent branched MAPs shown in FIG. 6A: A.beta. (3-10)(7-14) (Construct No. 8), A.beta. (1-8)(21-28) (Construct No. 11), A.beta. (3-10) (21-28) (Construct No-12).

[0091] Rhesus macaques (N=3) were immunized with 5 .mu.g of each of the vaccine formulated in Merck alum adjuvant (MAA) plus 100 ug of ISCOMATRIX.RTM. every four weeks. Serum samples were collected four weeks following each injection and determined for A.beta. specific antibody responses by ELISA. Consistent with the results from the guinea pig studies, all conjugates were found to be immunogenic in monkeys. A.beta. specific antibody titers were detectable after single injections and further boosted after the subsequent injections. Generally for the conjugates tested, the peak titers were reached after the second or third immunization where geometric mean titers ranged from 25,000 to 500,000. These results confirm the finding that the A.beta. conjugates described herein are capable of eliciting an A.beta. specific antibody response.

D. Adjuvant Effect on Immunogenicity of Conjugate Vaccines in Rhesus Monkeys

[0092] An additional study was conducted in non-human primates, i.e. rhesus monkeys, to determine whether an A.beta. peptide-OMPC conjugate and a saponin-based adjuvant, such as ISCOMATRIX.RTM., can provide an improved immune response. Applicants used an A.beta. (1-18) peptide conjugated to OMPC as the model antigen. Rhesus monkeys (Macaca mulatta) were maintained in accordance with the institutional animal care protocols of Merck Research Laboratories and New Iberia Research Center (The University of Louisiana at Lafayette, New Iberia, La.).

[0093] Five groups of monkeys, three per group, were given the following A.beta. (1-18)-OMPC conjugates: (1) 5 .mu.g conjugate (based on peptide content) in alum, (2) 5 .mu.g conjugate in alum+100 .mu.g ISCOMATRIX.RTM., (3) 5 .mu.g conjugate in alum+50 mg ISCOMATRIX.RTM., (4), 30 .mu.g conjugate in alum, (2) 30 .mu.g conjugate in alum+100 .mu.g ISCOMATRIX.RTM.. The immunizations were carried out by intramuscular injections in 0.5 ml aliquots at weeks 0, 8 and 24 using tuberculin syringes (Becton-Dickinson, Franklin Lakes, N.J.). Serum samples were collected at four week intervals starting from week 0 (pre-bleed) and the tested for antibody titers against A.beta..sub.40 by ELISA, performed as described in the preceding example.

[0094] Interium analysis of this ongoing study demonstrated that at PD1 the monkeys receiving 5 mcg conjugate vaccine in alum failed to develop any detectable titers, while those receiving 30 .mu.g conjugate vaccine in alum developed low A.beta..sub.40 specific titers. All monkeys that received the alum plus ISCOMATRIX.RTM. formulation developed significant antibody titers. At PD2, both doses of immunogen in alum alone produced similar titer levels, whereas the cohorts receiving the alum plus ISCOMATRIX.RTM. developed 10-fold higher antibody titers relative to the alum alone condition. The results of this study confirmed that this A.beta. peptide-OMPC conjugate is immunogenic in non-human primates. The data further demonstrate that the efficacy of such a conjugate vaccine is significantly enhanced by a saponin-based adjuvant such as ISCOMATRIX.RTM..

Example 4

Immunoreactivity of Guinea Pig Polyclonal Sera

[0095] In order to demonstrate that the immune sera generated from the guinea pigs above (Example 3.B) following immunization with 8-mer KLH conjugates is relevant to human AD, a study was performed to evaluate the immunoreactivity of polyclonal sera from a guinea pig immunized with an A.beta. (3-10)-KLH immunogen. Four weeks following a second injection of this construct blood was collected from a representative guinea pig according to the following methodology.

[0096] Reactivity of the polyclonal sera was evaluated on human AD brain sections (BioChain Institute, Inc., Hayward, Calif.). Human brain sections were prepared by incubating at 60.degree. C. for thirty minutes followed by two five minute xylene washes at room temperature. Sections were subsequently immersed in 100% EtOH twice for five minute each followed by a five minute immersion in ddH2O, Sections were immersed for three minutes in 99% formic acid followed by a brief wash in ddH2O and a five minute immersion in phosphate buffered solution (PBS). Sections were then incubated with a peroxidase blocker for ten minutes followed by a five minute PBS wash. Sections were blocked by a ten minute exposure to 10% goat serum followed by a five minute wash with PBS. Sections were then incubated with diluted guinea pig sera at 4.degree. C. overnight or for one hour at room temperature. Following a five minute PBS wash, sections were incubated for ten minutes with diluted biotinylated goat anti-guinea pig IgG or biotinylated horse anti-mouse antibody (1 drop in 5 ml PBS). Sections were washed for five minutes in PBS and subsequently incubated with ABC solution (Vectorstain ABC kit; Vector Laboratories, Inc.) for thirty minutes. Sections were washed with PBS for five minutes. Sections were then stained with DAB (DakoCytomation) for five minutes and washed with dd H.sub.2O, Sections were then counterstained in hematoxylin for thirty seconds and dehydrated in graded EtOH and Xylenes (70% EtOH for five minutes, 80% EtOH for five minutes, 100% EtOH for five minutes and xylenes for five minutes). Sections were then cover-slipped and evaluated by light microscopy.

[0097] The immunogenic response produced by the A.beta. (3-10)-KLH conjugate produced an antibody response that was directed against human AD brain tissue. As shown in FIG. 4, this human brain section has extensive A.beta. deposition in a manner typical to that expected for human AD. While pre-immunized guinea pig sera demonstrates a lack of immunoreactivity when exposed to this tissue, positive immunoreactivity of sera from this same guinea pig is noted following two injections of the A.beta. (3-10)=-KLH construct. These data demonstrate that the immunogenicity found by ELISA, and illustrated in FIG. 3, contains a significant antibody response directed against human A.beta. found in this AD tissue. Thus, the results extend the unexpected finding of differential immunogenicity by some A.beta. fragments to further demonstrate that this response is directed in a manner consistent with therapeutic application.

Example 5

Identification of Immunogenic Fragments Lacking T-Cell Epitopes

[0098] To identify immunogenic fragments lacking a T-cell epitope for use in the invention herein, the following Enzyme-Linked ImmunoSpot (ELISpot) assay can be used as a method to assess T-cell responses to a particular antigen. Immunogen fragments possessing T-cell epitopes are identified by the presence of a dark spot on the surface of a white membrane; each spot indicates the presence of a T-cell that has secreted interferon gamma (IFN-.gamma.) in response to the antigen (i.e. immunogenic fragment). Those skilled in the art of vaccines and immunology are familiar with this assay, see, for example, Larsson et al., AIDS 3: 767-777, 1999, and Mwau et al., AIDS Research and Human Retroviruses 18: 611-618, 2002. A recent review can be found in A. E. Kalyuzhny, Methods Mol. Biol. 302: 15-31, 2005.

[0099] Applicants used peripheral blood monocytes (PBMCs) from rhesus macaques (New Iberia Research Center, The University of Louisiana at Lafayette, New Iberia, La.) for response to the peptides A131-40 (American Peptide Co., Sunnyvale, Calif.) (amino acid sequence DAEFREIDSGYEVHHQKLVFFAEDVG SNKGAIIGLMVGGVV) (SEQ ID NO: 78) and A.beta.1-20 (Synpep, Dublin, Calif.) (amino acid sequence DAEFRHDSG YEVHEIQKINFF) (SEQ ID NO: 79).

[0100] Purified monoclonal mouse anti-monkey IFN-.gamma. (clone MD-1, Cat No. CT 525, U-CyTech biosciences, Utrecht, The Netherlands) was diluted in phosphate buffered saline (PBS) with 1% penicillin and streptomycin sulfate (GIBCO.RTM. Penicillin-Streptomycin, Cat. No. 15140-122, Invitrogen, Carlsbad, Calif.), then added to 96-well HTS IP sterile plates (Cat. No. MSIPS4W10, Millipore, Billerica, Mass.), and incubated for greater than twelve hours at 4.degree. C. Plates were washed and R10 [RPMI medium 1640 (GIBCO.RTM. Cat. No. 11875-093), 10% Fetal bovine serum (HyClone SH30070.03, Logan, Utah), 0.1% 50 mM 2-Mercaptoethanol (GIBCO.RTM. Cat. No. 21985-023), 1% 1M HEPES Buffer (GIBCO.RTM. 15630-080), 1% 200 mM L-glutamine (GIBCO.RTM. Cat. No. 25030-081), 1% 100 mM MEM sodium pyruvate solution (GIBCO.RTM. Cat. No. 11360-070), 1% penicillin-streptomycin solution (GIBCO.RTM. Cat. No. 15140-122)] was added before incubation for at least two hours at 37.degree. C. PBMCs were centrifuged and re-suspended in R10. PBMCs were counted on a Z2 Coulter counter (Beckman Coulter, Fullerton, Calif.). Each well of the aspirated plate received either 0.4 .mu.g of A.beta.1-40, A.beta.1-20, PHA (phytohemagglutinin, Cat No. L-8902, Sigma, St. Louis, Mo., positive control), or diluted DMSO (Sigma, negative control); 400000 PBMCs were then added to each well. Plates were incubated for 18-24 hours at 37.degree. C. in a humid CO.sub.2 incubator. Plates were washed in PBS with 5% FBS and 0.005% Tween; biotin-conjugated anti-monkey IFN-.gamma. polyclonal antibodies (U-CyTech biosciences, Utrecht, The Netherlands) were diluted in the same media and added to each plate; plates were then incubated at 4.degree. C. for 18-24 hours. Streptavidin-AP (Cat. No. 13043E, BD PharMingen, Franklin Lakes, NT) was diluted in the same media and added to washed plates; plates were incubated at room temperature and in the dark for two hours. Filtered 1-Step NBT/BCIP Substrate (Pierce, Rockford, Ill., Cat. No. 34042) was added and a further incubation at room temperature in the dark for ten minutes was performed. After washing, plates were allowed to dry before being imaged with a CCD camera and the spots within each well were automatically counted by computer.

[0101] Applicants have established that spot forming cells per million PBMCs (SFC/10.sup.6 PBMCs) must exceed 55 and must exceed 4-fold the negative control to be defined as a positive result; failing to meet both these criteria defines a negative result. Rhesus macaques were vaccinated with either a MAP construct comprising A.beta. (3-10)/(21-28) (Construct No. 12, FIG. 6A) conjugated to OMPC or with both of two monomeric constructs, A.beta. (3-10) (SEQ ID NO: 69) and A.beta. (21-28) (SEQ ID NO: 73) conjugated to OMPC. Each macaque was assayed during the vaccination regimen at monthly intervals for three or four months; the highest signal ever recorded against either A.beta. 1-40 or A.beta.1-20 is only 18 SFC/10.sup.6 PBMCs, significantly below the 55 SFC/10.sup.6 PBMCs criterion. Thus, all resulted in a negative score, providing strong evidence that the vaccines did not elicit T-cell responses and, as such, did not include a T-cell epitope.

Example 6

Elevation of Plasma A.beta.

[0102] Rhesus macaque non-human primates (N=3) were immunized with 5 .mu.g of the MAP A.beta. (3-10)/(21-28) conjugate (Construct No. 12, FIG. 6A) or its monomeric constituent conjugate, A.beta. (3-10) (SEQ ID NO: 69) and A.beta. (21-28) (SEQ ID NO: 73) linked to OMPC as the carrier and formulated in MAA plus 100 .mu.g ISCOMATRIX.RTM.. The rhesus primates received vaccinations every four weeks with bleeds collected and analyzed four weeks following each injection. Anti-A.beta..sub.40 titers and total A.beta..sub.1-40 levels were determined.

[0103] Plasma A.beta..sub.1-40 levels were determined in these immunized animals using a 6E10/G210 FT ISA. This assay measures A.beta..sub.1-40 using a sandwich ELISA comprising an N-terminal capture antibody 6E10 (A.beta. 1-8) (Signet Laboratories, Dedham, Mass.) and a C-terminal A.beta..sub.40 neo-epitope antibody (G210) (The Genetics Company, Inc., Zurich, Switzerland) conjugated with alkaline phosphatase. The antibody, 6E10, was coated onto plates at a concentration of 5 ug/ml. Diluted plasma samples (1:3) were used at 50 .mu.l/well in triplicates. A.beta..sub.1-40 standards were prepared from 400 pM-3 pM in 6E10 immuno-depleted rhesus plasma matrix. This assay has a signal-to-noise ratio of about 5-20. The CDP-star alkaline phosphatase substrate was obtained from Applied Biosystems, Foster City, Calif. SuperBlock.RTM., a pre-formulated blocking buffer, was obtained from Pierce Biotechnology, Rockford, Ill. (Cat#37515). Counts from individual samples, run in triplicate, were converted to actual analyte concentrations using a third order spline fit to the standards. QC samples were run to evaluate plate to plate variability of the signal.

[0104] As depicted in FIG. 8A, the results of these analyses demonstrated a greater than 3-fold increase in plasma A.beta..sub.40 at PD3 following immunization with the MAP A.beta. (3-10)/(21-28) construct (Construct No. 12, FIG. 6A). This increase in plasma A.beta..sub.40 was not observed in animals immunized with the monomeric A.beta. conjugate/OMPC vaccine constructs. Specifically, immunization using either A.beta. (3-10) (SEQ ID NO: 69) or A.beta. (21-28) (SEQ ID NO: 73) produced a lack of, or appreciably lower, response on this measure. It was notable that these differences were independent of titer levels as depicted in FIG. 8B.

[0105] Collectively, these data demonstrate that some constructs have an advantage relative to other immunogenic constructs with respect to their ability to elevate plasma A.beta. levels. Those skilled in the art would appreciate that this selectivity of immunogenic fragments, i.e. the ability to elevate plasma A.beta. levels, has not been shown prior to the invention herein and was not predictable from the prior art. As such, the identification of immunogens, either 8-mers or MAPs, lacking a T-cell epitope, that elevate plasma A.beta. following immunization, provides a method for selecting said 8-mers or MAPs for use in a vaccine construct. As a result of the invention herein, those skilled in the art are now able to characterize said vaccine constructs both quantitatively (i.e., immunogenicity) and qualitatively (i.e., nature of the polyclonal antibody response--ability to elevate plasma AB levels). It will be further appreciated by those skilled in the art that the invention herein is not limited to 8-amino acid A.beta. fragments, but is inclusive of any antigen capable of producing a polyclonal antibody response in the host organism that is reactive to A.beta..

Sequence CWU 1

1

79142PRTArtificial SequenceSynthetic Peptide 1Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val Ile Ala 35 4028PRTArtificial SequenceSynthetic Peptide 2Asp Ala Glu Phe Arg His Asp Ser1 538PRTArtificial SequenceSynthetic Peptide 3Ala Glu Phe Arg His Asp Ser Gly1 548PRTArtificial SequenceSynthetic Peptide 4Glu Phe Arg His Asp Ser Gly Tyr1 558PRTArtificial SequenceSynthetic Peptide 5Phe Arg His Asp Ser Gly Tyr Glu1 568PRTArtificial SequenceSynthetic Peptide 6Arg His Asp Ser Gly Tyr Glu Val1 578PRTArtificial SequenceSynthetic Peptide 7His Asp Ser Gly Tyr Glu Val His1 588PRTArtificial SequenceSynthetic Peptide 8Asp Ser Gly Tyr Glu Val His His1 598PRTArtificial SequenceSynthetic Peptide 9Ser Gly Tyr Glu Val His His Gln1 5108PRTArtificial SequenceSynthetic Peptide 10Gly Tyr Glu Val His His Gln Lys1 5118PRTArtificial SequenceSynthetic Peptide 11Tyr Glu Val His His Gln Lys Leu1 5128PRTArtificial SequenceSynthetic Peptide 12Glu Val His His Gln Lys Leu Val1 5138PRTArtificial SequenceSynthetic Peptide 13Val His His Gln Lys Leu Val Phe1 5148PRTArtificial SequenceSynthetic Peptide 14His His Gln Lys Leu Val Phe Phe1 5158PRTArtificial SequenceSynthetic Peptide 15His Gln Lys Leu Val Phe Phe Ala1 5168PRTArtificial SequenceSynthetic Peptide 16Gln Lys Leu Val Phe Phe Ala Glu1 5178PRTArtificial SequenceSynthetic Peptide 17Lys Leu Val Phe Phe Ala Glu Asp1 5188PRTArtificial SequenceSynthetic Peptide 18Leu Val Phe Phe Ala Glu Asp Val1 5198PRTArtificial SequenceSynthetic Peptide 19Val Phe Phe Ala Glu Asp Val Gly1 5208PRTArtificial SequenceSynthetic Peptide 20Phe Phe Ala Glu Asp Val Gly Ser1 5218PRTArtificial SequenceSynthetic Peptide 21Phe Ala Glu Asp Val Gly Ser Asn1 5228PRTArtificial SequenceSynthetic Peptide 22Ala Glu Asp Val Gly Ser Asn Lys1 5238PRTArtificial SequenceSynthetic Peptide 23Glu Asp Val Gly Ser Asn Lys Gly1 5248PRTArtificial SequenceSynthetic Peptide 24Asp Val Gly Ser Asn Lys Gly Ala1 5258PRTArtificial SequenceSynthetic Peptide 25Val Gly Ser Asn Lys Gly Ala Ile1 5268PRTArtificial SequenceSynthetic Peptide 26Gly Ser Asn Lys Gly Ala Ile Ile1 5278PRTArtificial SequenceSynthetic Peptide 27Ser Asn Lys Gly Ala Ile Ile Gly1 5288PRTArtificial SequenceSynthetic Peptide 28Asn Lys Gly Ala Ile Ile Gly Leu1 5298PRTArtificial SequenceSynthetic Peptide 29Lys Gly Ala Ile Ile Gly Leu Met1 5308PRTArtificial SequenceSynthetic Peptide 30Gly Ala Ile Ile Gly Leu Met Val1 5318PRTArtificial SequenceSynthetic Peptide 31Ala Ile Ile Gly Leu Met Val Gly1 5328PRTArtificial SequenceSynthetic Peptide 32Ile Ile Gly Leu Met Val Gly Gly1 5338PRTArtificial SequenceSynthetic Peptide 33Ile Gly Leu Met Val Gly Gly Val1 5348PRTArtificial SequenceSynthetic Peptide 34Gly Leu Met Val Gly Gly Val Val1 5358PRTArtificial SequenceSynthetic Peptide 35Leu Met Val Gly Gly Val Val Ile1 5368PRTArtificial SequenceSynthetic Peptide 36Met Val Gly Gly Val Val Ile Ala1 53718PRTArtificial SequenceSynthetic Peptide 37Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val388PRTArtificial SequenceSynthetic Peptide 38Asp Ala Glu Phe Arg His Asp Ser1 5398PRTArtificial SequenceSynthetic Peptide 39Ala Glu Phe Arg His Asp Ser Gly1 5408PRTArtificial SequenceSynthetic Peptide 40Glu Phe Arg His Asp Ser Gly Tyr1 5418PRTArtificial SequenceSynthetic Peptide 41Phe Arg His Asp Ser Gly Tyr Glu1 5428PRTArtificial SequenceSynthetic Peptide 42Arg His Asp Ser Gly Tyr Glu Val1 5438PRTArtificial SequenceSynthetic Peptide 43His Asp Ser Gly Tyr Glu Val His1 5448PRTArtificial SequenceSynthetic Peptide 44Asp Ser Gly Tyr Glu Val His His1 5458PRTArtificial SequenceSynthetic Peptide 45Ser Gly Tyr Glu Val His His Gln1 5468PRTArtificial SequenceSynthetic Peptide 46Gly Tyr Glu Val His His Gln Lys1 5478PRTArtificial SequenceSynthetic Peptide 47Tyr Glu Val His His Gln Lys Leu1 5488PRTArtificial SequenceSynthetic Peptide 48Glu Val His His Gln Lys Leu Val1 5498PRTArtificial SequenceSynthetic Peptide 49His His Gln Lys Leu Val Phe Phe1 55011PRTArtificial SequenceSynthetic Peptide 50Glu Val His His Gln Lys Leu Val Glu Glu Glu1 5 105111PRTArtificial SequenceSynthetic Peptide 51His His Gln Lys Leu Val Phe Phe Glu Glu Glu1 5 105211PRTArtificial SequenceSynthetic Peptide 52His His Gln Lys Leu Val Phe Phe Lys Lys Lys1 5 105311PRTArtificial SequenceSynthetic Peptide 53Gln Lys Leu Val Phe Phe Ala Glu Lys Lys Lys1 5 105411PRTArtificial SequenceSynthetic Peptide 54Leu Val Phe Phe Ala Glu Asp Val Lys Lys Lys1 5 10558PRTArtificial SequenceSynthetic Peptide 55Leu Val Phe Phe Ala Glu Asp Val1 55611PRTArtificial SequenceSynthetic Peptide 56Phe Phe Ala Glu Asp Val Gly Ser Lys Lys Lys1 5 10578PRTArtificial SequenceSynthetic Peptide 57Ala Glu Asp Val Gly Ser Asn Lys1 5588PRTArtificial SequenceSynthetic Peptide 58Asp Val Gly Ser Asn Lys Gly Ala1 55911PRTArtificial SequenceSynthetic Peptide 59Gly Ser Asn Lys Gly Ala Ile Ile Lys Lys Lys1 5 106011PRTArtificial SequenceSynthetic Peptide 60Asn Lys Gly Ala Ile Ile Gly Leu Lys Lys Lys1 5 106111PRTArtificial SequenceSynthetic Peptide 61Gly Ala Ile Ile Gly Leu Met Val Glu Glu Glu1 5 106211PRTArtificial SequenceSynthetic Peptide 62Ile Ile Gly Leu Met Val Gly Gly Lys Lys Lys1 5 10638PRTArtificial SequenceSynthetic Peptide 63Gly Leu Met Val Gly Gly Val Val1 56411PRTArtificial SequenceSynthetic Peptide 64Gly Leu Met Val Gly Gly Val Val Lys Lys Lys1 5 106511PRTArtificial SequenceSynthetic Peptide 65Met Val Gly Gly Val Val Ile Ala Lys Lys Lys1 5 106642PRTArtificial SequenceSynthetic Peptide 66Glu Val Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40678PRTArtificial SequenceSynthetic Peptide 67Asp Ala Glu Phe Arg His Asp Ser1 5688PRTArtificial SequenceSynthetic Peptide 68Ala Glu Phe Arg His Asp Ser Gly1 5698PRTArtificial SequenceSynthetic Peptide 69Glu Phe Arg His Asp Ser Gly Tyr1 5708PRTArtificial SequenceSynthetic Peptide 70Asp Ser Gly Tyr Glu Val His His1 5718PRTArtificial SequenceSynthetic Peptide 71Ser Gly Tyr Glu Val His His Gln1 5728PRTArtificial SequenceSynthetic Peptide 72Leu Val Phe Phe Ala Glu Asp Val1 5738PRTArtificial SequenceSynthetic Peptide 73Ala Glu Asp Val Gly Ser Asn Lys1 5748PRTArtificial SequenceSynthetic Peptide 74Gly Leu Met Val Gly Gly Val Val1 57518PRTArtificial SequenceSynthetic Peptide 75Glu Val Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val7610PRTArtificial SequenceSynthetic Peptide 76Glu Val Glu Phe Arg His Asp Ser Gly Tyr1 5 10778PRTArtificial SequenceSynthetic Peptide 77Glu Val Glu Phe Arg His Asp Ser1 57840PRTArtificial SequenceSynthetic Peptide 78Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val 35 407920PRTArtificial SequenceSynthetic Peptide 79Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe 20

Claims

1-18. (canceled)

19. A method for preventing or treating a disease associated with amyloid deposits of A.beta. in the brain of a patient comprising administering an effective dose of a composition comprising a multivalent branched multiple antigen peptide (MAP) linked to a carrier to form a conjugate, wherein the MAP comprises two or more non-identical immunogenic linear 8 amino acid peptide fragments (8-mers) of A.beta., each fragment lacking a T-cell epitope, and wherein one of said 8-mers is A.beta. 21-28 (AEDVGSNK) (SEQ ID NO: 22).

20. The method of claim 19 wherein one of the immunogenic fragments of A.beta. is selected from the group consisting of an 8-mer corresponding to amino acid regions A.beta. 1-8, A.beta. 2-9, A.beta. 3-10, A.beta. 7-14, A.beta. 17-24, A.beta. 21-28, and A.beta. 33-40.

21. The method of claim 19 wherein the MAP is selected from the group consisting of amino acid regions a) A.beta. 1-8 and A.beta. 21-28, b) A.beta. 3-10 and A.beta. 21-28, c) A.beta. 7-14 and A.beta. 21-28 and A.beta. 3-10, and d) A.beta. 7-14 and A.beta. 33-40 and A.beta. 21-28 and A.beta. 3-10, wherein each of the 8-mers are linked together.

22. The method of claim 21 wherein the MAP comprises the 8-mers A.beta. 3-10 and A.beta. 21-28 linked together on a lysine-based scaffold.

23. The method of claim 19 wherein the carrier is selected from the group consisting of serum albumin, keyhole limpet hemocyanin (KLH), an immunoglobulin, a tetanus toxoid protein, a bacterial toxoid protein, and an attenuated toxin derivative.

24. The method of claim 22 wherein the carrier is the outer membrane protein complex of Neisseria meningitidis (OMPC).

25. A method for preventing or treating a disease associated with amyloid deposits of A.beta. in the brain of a patient, comprising administering an effective dose of a pharmaceutical composition, comprising the MAP of claim 19, and a pharmaceutically acceptable adjuvant.

26. The method of claim 25 wherein the pharmaceutically acceptable adjuvant is selected from the group consisting of an aluminum salt, alum, a lipid, and a saponin-based adjuvant.

27. A method for preventing or treating a disease associated with amyloid deposits of A.beta. in the brain of a patient comprising administering an effective dose of a pharmaceutical composition comprising a MAP linked to a carrier to form a conjugate, wherein the MAP comprises the 8-mers A.beta. 3-10 and A.beta. 21-28 linked together on a lysine-based scaffold and the carrier is OMPC, and a saponin-based adjuvant.