Synthesis, solution conformation, and antibody recognition of oligotuftsin-based conjugates containing a beta-amyloid(4-10) plaque-specific epitope

One possible therapeutic approach to treat or prevent Alzheimer's disease (AD) is immunotherapy. On the basis of the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, conjugates with defined structures containing the epitope peptide attached to a tetratuftsin derivative as an oligopeptide carrier were synthesized and their structure characterized. To produce immunogenic constructs, the Abeta(4-10) epitope alone or flanked by alpha- or beta-alanine residues was attached through an amide bond to the tetratuftsin derivative (Ac-[TKPKG]4-NH2) or to a carrier peptide elongated by a promiscuous T-helper cell epitope (Ac-FFLLTRILTIPQSLD-[TKPKG]4-NH2). The conformational preferences of the carrier and conjugates were examined by CD spectroscopy in water and in 1:1 and 9:1 TFE:water mixtures (v/v). We found that the presence of flanking dimers in the conjugates had no effects on the generally unordered solution conformation of the conjugates. However, conjugates with an elongated peptide backbone exhibited CD spectra indicative for a partially ordered secondary structure in the presence of TFE. Comparative ELISA binding studies, using monoclonal antibody raised against the beta-amyloid (1-17) peptide, showed that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Abeta(4-10) B-cell epitope with the beta-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Abeta with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.     

Design, structural, and immuno-analytical properties of antigenic bioconjugates comprising a beta-amyloid-plaque specific epitope

Immunotherapeutic approaches are investigated for treatment of neurodegenerative diseases of the Alzheimer's dementia (AD) type. The identification of a beta-amyloid-plaque specific epitope, Abeta(4-10) (4FRHDSGY10), recognized by therapeutically active antibodies from transgenic AD mice could provide the basis for the development of AD vaccines. Here we report on the synthesis, structural and immuno-analytical characterization of bioconjugates comprising the beta-amyloid(4-10) epitope as new vaccine lead structures against Alzheimer's disease. To produce antigenic bioconjugates, potential immunogens, the epitope peptide elongated by a cysteine residue or a cysteinyl-pentaglycine hexapeptide unit either at the N- or C-terminus was attached via a thioether bond to synthetic oligopeptide carriers, such as oligotuftsin derivatives, sequential oligopeptide carrier, or lysine dendrimer. The antigenic properties of these constructs were determined by enzyme-linked immunosorbent assay (ELISA) using an anti-Abeta(1-17) monoclonal antibody. Our results indicate that the major factors which influence the antibody binding of the Abeta(4-10) epitope are (i) the epitope topology and (ii) the presence of a spacer moiety between the carrier and the epitope peptide. Interestingly, the carrier type had no marked effect on the binding of the antibody to the epitope-conjugates. The conformational preferences of the conjugates were examined by circular dichroism spectroscopy in water and in trifluoroethanol. In water, the conjugates adopt random coil conformation independently on their primary structure. However, differences related to the attachment site of the epitope to the carriers were determined in TFE, conjugates in which the epitope was attached to the carrier through the N-terminus exhibiting more ordered secondary structure.     

Synthesis and comparison of antibody recognition of conjugates containing herpes simplex virus type 1 glycoprotein D epitope VII

Synthetic oligopeptides comprising linear or continuous topographic B-cell epitope sequences of proteins might be considered as specific and small size antigens. It has been demonstrated that the strength and specificity of antibody binding could be altered by conjugation to macromolecules or by modification in the flanking regions. However, no systematic studies have been reported to describe the effect of different carrier macromolecules in epitope conjugates. To this end, the influence of carrier structure and topology on antibody recognition of attached epitope has been studied by comparing the antibody binding properties of a new set of conjugates with tetratuftsin analogue (H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2), T20) sequential oligopeptide carrier (SOC(n)), branched chain polypeptide, poly[Lys(Ser(i)-DL-Ala(m))] (SAK), multiple antigenic peptide (MAP), and keyhole limpet hemocyanine (KLH). In these novel constructs, peptide (9)LKNleADPNRFRGKDL(22) ([Nle(11)]-9-22) representing an immunodominant B cell epitope of herpes simplex virus type 1 glycoprotein D (HSV-1 gD) was conjugated to polypeptides through a thioether or amide bond. Here we report on the preparation of sequential and polymeric polypeptides possessing chloroacetyl groups in multiple copies at the alpha- and/or epsilon-amino group of the polypeptides and its use for the conjugation of epitope peptides possessing Cys at C-terminal position. We have performed binding studies (direct and competitive ELISA) with monoclonal antibody (Mab) A16, recognizing the HSV gD-related epitope, [Nle(11)]-9-22, and conjugates containing identical and uniformly oriented epitope peptide in multiple copies attached to five different macromolecules as carrier. Data suggest that the chemical nature of the carrier and the degree of substitution have marked influence on the strength of antibody binding.     

Conformation-sensitive antibodies against alzheimer amyloid-beta by immunization with a thioredoxin-constrained B-cell epitope peptide

Immunotherapy against the amyloid-beta (Abeta) peptide is a valuable potential treatment for Alzheimer disease (AD). An ideal antigen should be soluble and nontoxic, avoid the C-terminally located T-cell epitope of Abeta, and yet be capable of eliciting antibodies that recognize Abeta fibrils and neurotoxic Abeta oligomers but not the physiological monomeric species of Abeta. We have described here the construction and immunological characterization of a recombinant antigen with these features obtained by tandem multimerization of the immunodominant B-cell epitope peptide Abeta1-15 (Abeta15) within the active site loop of bacterial thioredoxin (Trx). Chimeric Trx(Abeta15)n polypeptides bearing one, four, or eight copies of Abeta15 were constructed and injected into mice in combination with alum, an adjuvant approved for human use. All three polypeptides were found to be immunogenic, yet eliciting antibodies with distinct recognition specificities. The anti-Trx(Abeta15)4 antibody, in particular, recognized Abeta42 fibrils and oligomers but not monomers and exhibited the same kind of conformational selectivity against transthyretin, an amyloidogenic protein unrelated in sequence to Abeta. We have also demonstrated that anti-Trx(Abeta15)4, which binds to human AD plaques, markedly reduces Abeta pathology in transgenic AD mice. The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained Abeta fragment repeat and identify Trx(Abeta15)4 as a promising new tool for AD immunotherapy.     

Duration and specificity of humoral immune responses in mice vaccinated with the Alzheimer's disease-associated beta-amyloid 1-42 peptide.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by overproduction of beta-amyloid (Abeta), which is formed from amyloid precursor protein (APP), with the subsequent pathologic deposition of Abeta in regions of the brain important for memory and cognition. Recently, vaccination of murine models of AD that exhibit Abeta deposition has halted or delayed the usual progression of the pathology of AD. Our group has demonstrated that vaccination of a doubly transgenic mouse model (expressing mutant APP and presenilin-1) with the Abeta 1-42 peptide protects these mice from the memory deficits they would ordinarily develop. This report further characterizes the Abeta 1-42 peptide vaccine in mice. Anti-Abeta response time course analysis indicated that at least three vaccinations (each 100 microg) were necessary to elicit a significant anti-Abeta titer. Subsequent vaccinations resulted in half-maximal antibody titers of at least 10,000, and these titers were maintained for at least 5 months after the final boost. Peptide binding competition studies indicated that the highest humoral responses are generated against the N terminus of the Abeta peptide. Also, measurement of specific murine Ig isotypes in Abeta-vaccinated mice demonstrated a predominant IgG(1) and IgG(2b) response, suggesting a type 2 (Th2) T-helper cell immune response, which drives humoral immunity. Finally, lymphocyte proliferation assay experiments using Abeta peptides and splenocytes from vaccinated mice demonstrated that the vaccine specifically stimulates T-cell epitopes present within the Abeta peptide.     

Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide

Immunization of amyloid precursor protein transgenic mice with fibrillar beta-amyloid (Abeta) prevents Alzheimer's disease (AD)-like neuropathology. The first immunotherapy clinical trial used fibrillar Abeta, containing the B and T cell self epitopes of Abeta, as the immunogen formulated with QS21 as the adjuvant in the vaccine. Unfortunately, the clinical trial was halted during the phase II stage when 6% of the participants developed meningoencephalitis. The cause of the meningoencephalitis in the patients that received the vaccine has not been definitively determined; however, analysis of two case reports from the AN-1792 vaccine trial suggest that the meningoencephalitis may have been caused by a T cell-mediated autoimmune response, whereas production of anti-Abeta Abs may have been therapeutic to the AD patients. Therefore, to reduce the risk of an adverse T cell-mediated immune response to Abeta immunotherapy we have designed a prototype epitope vaccine that contains the immunodominant B cell epitope of Abeta in tandem with the synthetic universal Th cell pan HLA DR epitope, pan HLA DR-binding peptide (PADRE). Importantly, the PADRE-Abeta(1-15) sequence lacks the T cell epitope of Abeta. Immunization of BALB/c mice with the PADRE-Abeta(1-15) epitope vaccine produced high titers of anti-Abeta Abs. Splenocytes from immunized mice showed robust T cell stimulation in response to peptides containing PADRE. However, splenocytes from immunized mice were not reactivated by the Abeta peptide. New preclinical trials in amyloid precursor protein transgenic mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse events that occurred in the first clinical trial.     

Phospholipid-model membrane interactions with branched polypeptide conjugates of a hepatitis A virus peptide epitope

To establish correlation between structural properties (charge, composition, and conformation) and membrane penetration capability, the interaction of epitope peptide-carrier constructs with phospholipid model membranes was studied. For this we have conjugated a linear epitope peptide, (110)FWRGDLVFDFQV(121) (110-121), from VP3 capside protein of the Hepatitis A virus with polylysine-based branched polypeptides with different chemical characteristics. The epitope peptide elongated by one Cys residue at the N-terminal [C(110-121)] was attached to poly[Lys-(DL-Ala(m)()-X(i)())] (i < 1, m approximately 3), where x = ?(AK), Ser (SAK), or Glu (EAK) by the amide-thiol heterobifunctional reagent, 3-(2-pyridyldithio)propionic acid N-hydroxy-succinimide ester. The interaction of these polymer-[C(110-121)] conjugates with phospholipid monolayers and bilayers was studied using DPPC and DPPC/PG (95/5 mol/mol) mixture. Changes in the fluidity of liposomes induced by these conjugates were detected by using two fluorescent probes 1,6-diphenyl-1,3, 5-hexatriene (DPH) and sodium anilino naphthalene sulfonate (ANS). The binding of conjugates to the model membranes was compared and the contribution of the polymer component to these interactions were evaluated. We found that conjugates with polyanionic/EAK-[C(110-121)] or polycationic/SAK-[C(110-121)], AK-[C(110-121)]/character were capable to form monomolecular layers at the air/water interface with structure dependent stability in the following order: EAK-[C(110-121)] > SAK-[C(110-121)] > AK-[C(110-121)]. Data obtained from penetration studies into phospholipid monolayers indicated that conjugate insertion is more pronounced for EAK-[C(110-121)] than for AK-[C(110-121)] or SAK-[C(110-121)]. Changes in the fluorescence intensity and in polarization of fluorescent probes either at the polar surface (ANS) or within the hydrophobic core (DPH) of the DPPC/PG liposomes suggested that all three conjugates interact with the outer surface of the bilayer. Marked penetration was documented by a significant increase of the transition temperature only with the polyanionic compound/EAK-[C(110-121)]. Taken together, we found that the binding/penetration of conjugates to phospholipid model membranes is dependent on the charge properties of the constructs. Considering that the orientation and number of VP3 epitope peptides attached to branched polypeptides were almost identical, we can conclude that the structural characteristics (amino acid composition, charge, and surface activity) of the carrier have a pronounced effect on the conjugate-phospholipid membrane interaction. These observations suggest that the selection of polymer carrier for epitope attachment might significantly influence the membrane activity of the conjugate and provide guidelines for adequate presentation of immunogenic peptides to the cells.     

A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response.

A solid-phase 8-mer random combinatorial peptide library was used to generate a panel of mimotopes of an epitope recognized by a monoclonal antibody to the F protein of measles virus (MV). An inhibition immunoassay was used to show that these peptides were bound by the monoclonal antibody with different affinities. BALB/c mice were coimmunized with the individual mimotopes and a T-helper epitope peptide (from MV fusion protein), and the reactivity of the induced anti-mimotope antibodies with the corresponding peptides and with MV was determined. The affinities of the antibodies with the homologous peptides ranged from 8.9 x 10(5) to 4.4 x 10(7) liters/mol. However, only one of the anti-mimotope antibodies cross-reacted with MV in an enzyme-linked immunosorbent assay and inhibited MV plaque formation. Coimmunization of mice with this mimotope and the T-helper epitope peptide induced an antibody response which conferred protection against fatal encephalitis induced following challenge with MV and with the structurally related canine distemper virus. These results indicate that peptide libraries can be used to identify mimotopes of conformational epitopes and that appropriate immunization with these mimotopes can induce protective antibody responses.     

Papillomavirus-like particles are an effective platform for amyloid-beta immunization in rabbits and transgenic mice

Immunization with amyloid-beta (Abeta) prevents the deposition of Abeta in the brain and memory deficits in transgenic mouse models of Alzheimer's disease (AD), opening the possibility for immunotherapy of AD in humans. Unfortunately, the first human trial of Abeta vaccination was complicated, in a small number of vaccinees, by cell-mediated meningoencephalitis. To develop an Abeta vaccine that lacks the potential to induce autoimmune encephalitis, we have generated papillomavirus-like particles (VLP) that display 1-9 aa of Abeta protein repetitively on the viral capsid surface (Abeta-VLP). This Abeta peptide was chosen because it contains a functional B cell epitope, but lacks known T cell epitopes. Rabbit and mouse vaccinations with Abeta-VLP were well tolerated and induced high-titer autoAb against Abeta, that inhibited effectively assembly of Abeta(1-42) peptides into neurotoxic fibrils in vitro. Following Abeta-VLP immunizations of APP/presenilin 1 transgenic mice, a model for human AD, we observed trends for reduced Abeta deposits in the brain and increased numbers of activated microglia. Furthermore, Abeta-VLP vaccinated mice also showed increased levels of Abeta in plasma, suggesting efflux from the brain into the vascular compartment. These results indicate that the Abeta-VLP vaccine induces an effective humoral immune response to Abeta and may thus form a basis to develop a safe and efficient immunotherapy for human AD.     

Toward a carbohydrate-based HIV-1 vaccine: synthesis and immunological studies of oligomannose-containing glycoconjugates

Human antibody 2G12 is a broadly neutralizing antibody that exerts its anti-HIV activity by targeting a novel oligomannose cluster on HIV-1 gp120. It was previously demonstrated that synthetic oligomannose clusters could mimic the carbohydrate epitope of 2G12 and showed enhanced antigenicity (Wang L. X. et al. (2004) Chem.Biol. 11, 127). This paper describes the synthesis of oligomannose-containing glycoconjugates that include either a carrier protein or a universal T-helper epitope peptide to provide an effective immunogen. It was shown that the synthetic neoglycoconjugates containing oligomannose clusters could be recognized by the human antibody 2G12. Rabbit immunization studies revealed that only a small fraction of antibodies raised by the glycoconjugates was directed to the carbohydrate antigens, with the majority of the IgG type antibodies being directed to the linkers in the conjugates. The anti-sera showed weak cross-reactivity to HIV-1 gp120.     

Modulation of Alzheimer's pathology by cerebro-ventricular grafting of hybridoma cells expressing antibodies against Abeta in vivo

Accumulation in brain of the beta-amyloid peptide (Abeta) is considered as crucial pathogenic event causing Alzheimer's disease (AD). Anti-Abeta immune therapy is a powerful means for Abeta clearance from the brain. We recently showed that intravenous injections of anti-Abeta antibodies led to reduction, elevation or no change in brain Abeta42 concentrations of an AD mouse model. We report here, in a second passive immunization protocol, a different bioactivity of same antibodies to alter brain Abeta42 concentrations. Comparing the bioactivity of anti-Abeta antibodies in these two passive immunization paradigms underscores the potential of immune therapy for AD treatment and suggests that both the epitope recognized by the antibody and the mode of antibody administration are crucial for its biological activity.     

Immunotherapeutic strategies for prevention and treatment of Alzheimer's disease.

Pathologic examination in Alzheimer's disease (AD) shows a significant correlation between beta-amyloid peptide (AbetaP) deposition and the clinical severity of dementia. Formation of beta-amyloid (Abeta) is a complex kinetic and thermodynamic process, dependent on peptide-peptide interactions that may be modulated by other proteins. We found that site-directed antibodies toward peptide EFRH sequences 3-6 of the N-terminal region of AbetaP suppress in vitro formation of Abeta and dissolve already-formed fibrillar amyloid. These so-called chaperone-like properties of monoclonal antibodies led to the development of a new immunologic approach to AD treatment. The immunization procedure, based on phages displaying the EFRH epitope as antigen, induced anti-AbetaP antibodies that recognized the whole AbetaP and exhibited antiaggregating properties similar to those of antibodies obtained by injection of Abeta fibrils. Production and performance of anti-beta-amyloid antibodies in the transgenic mouse model of AD showed that these antibodies may be delivered from the periphery to the central nervous system, preventing the formation of Abeta and dissolving already-present aggregates. Moreover, immunization with Abeta protected transgenic mice from the learning and age-related memory deficits that occur in AD. These data support the hypotheses that Abeta plays a central role in AD and that site-directed antibodies that modulate Abeta conformation may provide immunotherapy of the disease.     

Robust immune responses elicited by a fully synthetic three-component vaccine

The overexpression of saccharides such as Globo-H, Lewis(Y) and Tn antigen is a common feature of oncogenic transformed cells. Endeavors to exploit this aberrant glycosylation for cancer vaccine development have been complicated by difficulties in eliciting high titers of IgG antibodies against classical conjugates of tumor-associated carbohydrates to carrier proteins. We have designed, chemically synthesized and immunologically evaluated a number of fully synthetic vaccine candidates to establish strategies to overcome the poor immunogenicity of tumor-associated carbohydrates and glycopeptides. We have found that a three-component vaccine composed of a TLR2 agonist, a promiscuous peptide T-helper epitope and a tumor-associated glycopeptide can elicit in mice exceptionally high titers of IgG antibodies that can recognize cancer cells expressing the tumor-associated carbohydrate. The superior properties of the vaccine candidate are attributed to the local production of cytokines, upregulation of co-stimulatory proteins, enhanced uptake by macrophages and dendritic cells and avoidance of epitope suppression.     

Peptide based vaccine design: synthesis and immunological characterization of branched polypeptide conjugates comprising the 276-284 immunodominant epitope of HSV-1 glycoprotein D.

The importance of the length and conjugation site of a protective epitope peptide (276SALLEDPVG284) from glycoprotein D of herpes simplex virus in branched polypeptide conjugates has been investigated. A new set of peptides, with a single attachment site and truncated sequences, was prepared. The immunogenicity of conjugates and the specificity of antibody responses elicited were investigated in BALB/c, C57/B1/6 and CBA mice. It was found that the covalent coupling of the peptide comprising the 276-284 sequence of gD through its Asp residue at position 281 did not influence the immunogenic properties of the epitope, while involvement of the side chain of Glu at position 280 almost completely abolished immunogenicity. These results clearly indicated that the conjugation site of the epitope peptide influenced the intensity and specificity of antibody responses. Comparison of the immunological properties of conjugates containing truncated gD peptides revealed the presence of two epitopes within the 276-284 region. One of the proposed epitopes is situated at the N-terminal (276-281) region, while the other is located at the C-terminal end of the sequence (279-284). Binding data demonstrated that some of the peptides comprising these epitopes induced gD-specific responses in their conjugated form and also elicited an immune response that conferred protection against lethal HSV-1 infection. The correlation of peptide- and gD-specific antibody responses with the protective effect of the immune response is discussed.     

Zinc binding agonist effect on the recognition of the beta-amyloid (4-10) epitope by anti-beta-amyloid antibodies

Amyloid plaques associated to Alzheimer's disease present a high content of zinc ions. We previously showed that the N-terminal region of the amyloid peptide Abeta constitutes an autonomous zinc-binding domain. This region encompasses the previously identified epitope Abeta(4-10) targeted by antibodies capable to reduce amyloid deposition, but the influence of Abeta/Zn binding on the epitope recognition remains unknown. We demonstrate here the effect of Zn2+ ions on the recognition of peptides sharing the sequence of the Abeta N-terminal domain, by two monoclonal antibodies recognizing the beta-amyloid(4-10) epitope. The presence of Zn2+, but not of other cations, increased the recognition of the (1-16) peptide, while it was without effect on the recognition of the (1-10) peptide. These findings show a zinc-induced conformational change of the (1-16)-N-terminal region of AP3, which results in a better accessibility of the Abeta(4-10) epitope to the anti-Abeta antibodies, and suggest a role of zinc in epitope-based vaccination approaches.     

Multivalent thioether-peptide conjugates: B cell tolerance of an anti-peptide immune response.

Antibodies which bind beta2-glycoprotein I (beta2GPI) are associated with antiphospholipid syndrome. Synthetic peptide mimotopes have been discovered which compete with beta2GPI for binding to selected anti-beta2GPI. A thiol-containing linker was attached to the N-terminus of two cyclic thioether peptide mimotopes, peptides 1a and 1b. The resulting peptides, with linker attached, were reacted with two different haloacetylated platforms to prepare four tetravalent peptide-platform conjugates to be tested as B cell toleragens. The linker-containing peptides were reacted with maleimide-derivatized keyhole limpet hemocyanin (KLH) to provide peptide-KLH conjugates. Peptides 1a and 1b were also modified by acylation with 3-(4'-hydroxyphenyl)propionic acid N-hydroxysuccinimidyl ester. The resulting hydroxyphenyl peptides were radioiodinated and used to measure anti-peptide antibody levels. The KLH conjugates were used to immunize mice to generate an anti-peptide immune response. The immunized mice were treated with the conjugates or saline solution and boosted with the appropriate peptide-KLH conjugate. Three of the four conjugates suppressed the formation of anti-peptide antibody. The stabilities of the conjugates in mouse serum were measured, and the relative stabilities did not correlate with ability to suppress antibody formation.     

Induction of high levels of epitope-specific antibodies by epitope/peptide candidate vaccines against human immunodeficiency virus type-1 (HIV-1)

To test the immunogenicity of GPGRAFY-epitope-based candidate vaccines, a peptide with four repetitive GPGRAFY epitopes, V3-P1 [C-(GPGRAFY)4], and a peptide (PND) of the principal neutralizing domain (V3 loop: amino acid 301-328: C-TRPNNNTRKSIRIQRGPGRAFYTIGKI) on gp120 were synthesized and covalently coupled to a carrier protein BSA. Immunization of BALB/c mice and New Zealand White Rabbits with these conjugate vaccines engendered strong antibody responses against the PND (mouse serum titer by 1:12,800-25,600; rabbit serum titer by 1:6,400-12,800). Interestingly, the V3-P1-BSA conjugates and the PND-BSA conjugates could induce high levels of GPGRAFY-epitope-specific antibodies in the mice and rabbits (mouse serum titer by 1:25,600; rabbit serum titer by 1:12,800-25,600), while a recombinant gp160 subunit vaccine induced a low level of GPGRAFY-epitope-specific antibodies (serum titer by 1:400-1,600 in mice and rabbits). To confirm the above results, GPGRAFY-epitope-specific antibodies were isolated from rabbit sera induced by V3-P1-BSA, PND-BSA conjugates and rgp160 vaccine. In fact, 23-38 and 13-22 microg epitope-specific antibodies per milliliter serum were isolated from rabbit sera induced by V3-P1-BSA and PND-BSA conjugate, respectively, while 1.34 microg epitope-specific antibodies per milliliter serum were identified in rabbit serum induced by rgp160 vaccine. In the control group, only 0.069 microg proteins per milliliter serum were found in pooled pre-immune serum (normal serum). These results from mouse and rabbit experiments indicate that epitope and peptide vaccines both induce high levels of GPGRAFY-epitope-specific antibodies in comparison with rgp160 subunit vaccine, suggesting that epitope/peptide vaccines may be a new strategy to induce protective activity.     

Globular amyloid beta-peptide oligomer - a homogenous and stable neuropathological protein in Alzheimer's disease

Amyloid beta-peptide (Abeta)(1-42) oligomers have recently been discussed as intermediate toxic species in Alzheimer's disease (AD) pathology. Here we describe a new and highly stable Abeta(1-42) oligomer species which can easily be prepared in vitro and is present in the brains of patients with AD and Abeta(1-42)-overproducing transgenic mice. Physicochemical characterization reveals a pure, highly water-soluble globular 60-kDa oligomer which we named 'Abeta(1-42) globulomer'. Our data indicate that Abeta(1-42) globulomer is a persistent structural entity formed independently of the fibrillar aggregation pathway. It is a potent antigen in mice and rabbits eliciting generation of Abeta(1-42) globulomer-specific antibodies that do not cross-react with amyloid precursor protein, Abeta(1-40) and Abeta(1-42) monomers and Abeta fibrils. Abeta(1-42) globulomer binds specifically to dendritic processes of neurons but not glia in hippocampal cell cultures and completely blocks long-term potentiation in rat hippocampal slices. Our data suggest that Abeta(1-42) globulomer represents a basic pathogenic structural principle also present to a minor extent in previously described oligomer preparations and that its formation is an early pathological event in AD. Selective neutralization of the Abeta globulomer structure epitope is expected to have a high potential for treatment of AD.     

Design and chemical synthesis of a magnetic resonance contrast agent with enhanced in vitro binding, high blood-brain barrier permeability, and in vivo targeting to Alzheimer's disease amyloid plaques

Molecular imaging is an important new direction in medical diagnosis; however, its success is dependent upon molecular probes that demonstrate selective tissue targeting. We report the design and chemical synthesis of a derivative of human amyloid-beta (Abeta) peptide that is capable of selectively targeting individual amyloid plaques in the brain of Alzheimer's disease transgenic mice after being intravenously injected. This derivative is based on the sequence of the first 30 amino acid residues of Abeta with asparagyl/glutamyl-4-aminobutane residues (N-4ab/Q-4ab) substituted at unique Asp and Glu positions and with Gd-DTPA-aminohexanoic acid covalently attached at the N-terminal Asp. The Gd[N-4ab/Q-4ab]Abeta30 peptide was homogeneous as shown by high-resolution analytical techniques with a mass of +/-4385 Da determined by electrospray ionization mass spectrometry. This diamine- and gadolinium-substituted derivative of Abeta is shown to have enhanced in vitro binding to Alzheimer's disease (AD) amyloid plaques and increased in vivo permeability at the blood-brain barrier because of the unique Asp/Glu substitutions. In addition, specific in vivo targeting to AD amyloid plaques is demonstrated throughout the brain of an APP, PS1 transgenic mouse after intravenous injection. Because of the magnetic resonance (MR) imaging contrast enhancement provided by gadolinium, this derivative should enable the in vivo MR imaging of individual amyloid plaques in the brains of AD animals or patients to allow for early diagnosis and also provide a direct measure of the efficacy of anti-amyloid therapies currently being developed.     

The many faces of amyloid beta in Alzheimer's disease

The 'amyloid cascade hypothesis' links amyloid beta peptide (Abeta) with the pathological process of Alzheimer's disease (AD) and it still awaits universal acceptance. Amyloid precursor protein (APP), through the actions of the gamma-secretase complex, eventually becomes a different Abetaspecies. The various Abeta species have proven to be difficult to investigate under physiological conditions, and the species of Abeta responsible for neurotoxicity has yet to be unequivocally identified. The two important Abeta peptides involved are Abeta(1-40) and Abeta(1-42), and each has been ascribed both toxic and beneficial attributes. The ratio between the two species can be important in AD etiology. Additionally, shorter variants of Abeta peptides such as Abeta(1-8), Abeta(9-16) and Abeta(16) have also been shown to be potential participants in AD pathology. Interestingly, a new 56-kDa Abeta peptide (Abeta*56) disrupts memory when injected into the brains of young rats. Transgenic mice models are complicated by the interplay between various human Abeta types and the mouse Abeta types in the mouse brains. However, the accumulation of Abeta(1-42) in the brains of transgenic C. elegans worms and Drosophila is indeed detrimental. A less investigated aspect of AD is epigenetics, but in time the investigation of the role of epigenetics in AD may add to our understanding of the development of AD.