WO2009029272A2 - Epitope vaccine for prevention and reversion of ad pathology - Google Patents

Abstract

Alzheimer's disease (AD) that characterized by progressive cognitive decline, memory and behavior impairment, is the most common form of dementia in the elderly. A novel and potentially powerful strategy for AD prevention and treatment is an immunotherapy that via Abeta-specific antibodies could facilitate the clearance of amyloid deposits from the brains of AD patients. Epitope vaccines composed of several copies of self B cell epitope of amyloid peptide, foreign T cell epitope derived from the conventional vaccines or pathogens to which the human population is frequently exposed and molecular adjuvant are the subject of our patent. The rationale to use N>1 copies of B cell epitope in vaccine composition is to significantly enhance the anti-Aβ humoral immune response and the avidity of Aβ specific antibodies. The rationale to use foreign T cell epitope is to avoid the generation of autoreactive T cells and to overcome tolerance to self-epitope. More importantly, inclusion of various Th epitopes in the composition of the epitope vaccine will allow to induce quick and potent ant-Aβ antibody responses using pre-existing memory CD4+Th cells induced in general population by previous infections and/or immunizations with conventional vaccines, for example Tetanus, Diphtheria, Pertussis, Influenza, HBV.

Description

EPITOPE VACCINE FOR PREVENTION AND REVERSION OF AD PATHOLOGY

Cr oss-Reference to Related Applications

[001]This application claims the benefit of US Provisional Patent Application No. 60/966,218, filed August 27, 2007, and US Provisional Patent Application No. 61/124,274, filed April 16, 2008.

Field of the Invention

[002] The present invention relates to compositions and methods for generation of effective Alzheimer's disease (AD) vaccine.

Background of the Invention

[003]Alzheimer's disease (AD), characterized by progressive cognitive decline, memory and behavior impairment, and is the most common form of dementia in the elderly. It is estimated that there are currently about 18 million people worldwide with Alzheimer's disease. This number is projected to nearly double by 2025 to 34 million (http://who.org).

[004] The neuropathological features of the disease include neurofibrillary tangles (NFT), deposition of β-amyloid (Aβ) in senile plaques, and neuronal loss in affected brain regions(Price and Sisodia, 1994). These pathological changes result in a profound loss of neurons and synapses over the course of the disease, thereby contributing to a progressive reduction in the functional capacity of the patient. A critical aim in developing therapeutic interventions for AD is the identification of suitable targets. Aβ peptide is cleaved from the amyloid precursor protein (APP) by β- and γ-secretases (Esler and Wolfe, 2001 ; Selkoe, 1991 ; Selkoe, 1994) and is thought to play a central role in the onset and progression of AD which has led to the amyloid cascade hypothesis(Hardy and Selkoe, 2002; Hardy and Higgins, 1992). Of note, it is suggested that the most toxic forms of Aβ₄₂ peptide in brains are soluble oligomers of various size (Cleary et al., 2005; Gong et al., 2003; Klyubin et al., 2005; Lee et al., 2006; Lesne et al., 2006) and currently, there are no effective treatments for AD and new prophylactic and/or therapeutic approaches for treating AD are essential. Accordingly, different strategies currently being proposed as therapies for AD are aimed at reduction of the level of Aβ in the brain or blocking an assembly of the peptide into pathological forms (Gong et al., 2003; Klyubin et al., 2005; Lorenzo and Yankner, 1994) that disrupt cognitive functions (Cleary et al., 2005)and among them an immunotherapy.

[005]A novel and potentially powerful strategy for reducing the level of toxic forms of Aβ in the brain is an immunotherapy that via Aβ-specific antibodies could facilitate the clearance of amyloid deposits from the brains of APP transgenic mice (APP/Tg) and AD patients(Agadjanyan et al., 2005; Cribbs and Agadjanyan, 2005; Schenk et al., 2004; Weiner and Frenkel, 2006). In these studies both passive (Bard et al., 2000) and active (Janus et al., 2000; Morgan et al., 2000; Schenk et al., 1999) vaccination strategies have been very effective in mice and somewhat effective in humans(Ferrer et al., 2004; Fox et al., 2005; Gilman et al., 2005; Hock et al., 2003; Masliah et al., 2005; Nicoll et al., 2003). However, the first immunotherapy clinical trial on Alzheimer's patients, AN- 1792, was halted when a subset of those immunized with the vaccine containing the Aβ₄₂ formulated in QS21 developed adverse events (meningoencephalitis) in the central nervous system(Bayer et al., 2005; Ferrer et al., 2004; Fox et al., 2005; Gilman et al., 2005; Hock et al., 2003; Masliah et al., 2005; Nicoll et al., 2006; Nicoll et al., 2003; Orgogozo et al., 2003; Patton et al., 2006; Schenk, 2002; Weiner and Selkoe, 2002). While the actual cause of the adverse events is unknown, speculation has centered on autoreactive T-cells specific for the T-cell epitope in Aβ, the adjuvant (QS21), and the reformulation of the vaccine during the Phase Ha study(Schenk, 2002; Weiner and Selkoe, 2002). The low percentage of responders (-19%) and the generally low titers in the elderly patients in response to a self antigen (-1 :2200) even in the presence of a potent adjuvant (Bayer et al., 2005; Gilman et al., 2005; Patton et al., 2006) emphasizes the difficulty facing active immunization approaches in elderly AD patients with immunosenescence(Grubeck-Loebenstein and Wick, 2002).

[006] Recently a six-year follow-up studies of patients in AN 1792 trial showed that anti-Aβ antibodies can result in plaque removal from the AD brain. The extent of plaque removal is quite variable, but the extent of this process correlated to some extent with the titers of anti- Aβ antibodies in the serum of vaccinated AD patients. Importantly presence of plaques is not a prerequisite for progressive cognitive impairment in AD. Authors suggested that (i) the presence of Aβ plaques is required to initiate, but not to maintain the progressive neurodegeneration in AD; (ii) Amyloid plaques are an epiphenomenon, and extracellular soluble/oligomeric or intraneuronal forms of Aβ are responsible for the neurodegeneration in AD; (iii) using immunization as prevention rather than treatment will be more beneficial because it may block depositions of oligomeric forms of Aβ(Holmes et al., 2008).

[007] In an attempt to avoid the problems associated with active immunization of elderly AD patients other strategies have been proposed. One of them is the passive immunization strategy based on anti-Aβ antibody. Recently Elan Corporation, presented detailed results from their 18- month Phase 2 study with bapineuzumab (AAB-OOl) in patients with mild to moderate Alzheimer's disease (ICAD, 2008, Chicago, IL). Bapineuzumab is the first humanized monoclonal antibody designed to clear toxic Aβ from the brain. The double-blind, placebo- controlled multiple ascending dose trial was designed to assess the safety and tolerability of bapineuzumab in patients with mild-to-moderate Alzheimer's disease and to explore its efficacy at a range of doses. Statistically significant differences from baseline to week 78 were observed in favor of bapineuzumab treated ApoE4 non-carrier patients on both cognitive and functional efficacy endpoints. No statistically significant changes were observed in any of the cognitive or functional efficacy endpoints in the ApoE4 carrier patients. Adverse events (AE) were generally mild to moderate, but vasogenic edema detected in some patients appears to be dose related. Importantly, it has been shown that AN 1792 trial could increase cerebral amyloid angiopathy (CAA) as well and it was shown that passive immunotherapy increases CAA in transgenic mice and may causes increased incidence of microhemorrhage in vaccinated AD patients(Pfeifer et al., 2002; Racke et al., 2005; Wilcock et al., 2004). These data indicate that early treatment (before accumulation of toxic forms of amyloid in the vasculature) is necessary.

[008] Another strategy for generation of safe AD vaccine is attempt to avoid T cell responses specific to self-antigen, Aβ. For example, is the generation of an epitope vaccine composed of small B-cell epitope of Aβ₄₂ and irrelevant T helper (Th) cell epitope/s. Elan and Wyeth developed and are currently testing a conjugate vaccine (ACC-OOl, Phase 2), in which small N- terminal peptide fragments of Aβ are conjugated to a carrier protein derived from a non-toxic mutant of diphtheria toxin formulated in ThI -type adjuvant, QS21 (www.elan.com; http://www.alzforum.org/new/detail.asp?id=1807). [009] A potential problem for immunotherapy could be the fact that a reduction of insoluble Aβ may lead to increased levels of soluble forms of this peptide(Holmes et al., 2008; Patton et al., 2006), primarily oligomers, the most toxic form for neurons, and impair cognitive function(Haass and Selkoe, 2007). Our recent pre-clinical in vitro studies demonstrated that substoichiometric concentrations of purified anti-Aβ antibody prevented Aβ₄₂ aggregation and induced disaggregation of preformed Aβ₄₂ fibrils down to a non-filamentous and non-toxic species. However, anti-Aβ antibodies could not disaggregate oligomers although they did delay Aβ₄₂ oligomer formation(Mamikonyan et al., 2007). These in vitro observations suggest that therapeutic vaccination cannot disrupt in vivo toxic oligomers and may only minimally inhibit pre-existing AD pathology, therefore immunotherapy should be started before Aβ accumulation in the vasculature and parenchyma of brains induces an unalterable process. In other words, if AD pathology is associated with accumulation of oligomeric forms of Aβ₄₂ in the brain, the therapeutic vaccination strategy should not be effective. Instead protective vaccination should be used for effective prevention of AD pathology.

[0010] Recently, we reported about the efficacy of an epitope peptide vaccine composed of the immunodominant self-B cell epitope of Aβ₄₂ (Aβj.is or Aβi-π) in tandem with the strong non- self promiscuous T cell epitope. Data suggested that such vaccine could be safe and immunogenic in humans, because it (i) could bind with high affinity to 15 of 16 of the most common human HLA-DR molecules(Alexander et al., 2000; Alexander et al., 1998; La Rosa et al., 2002; Panina-Bordignon et al., 1989; Weber et al., 1999; Wei et al., 2001); (ii) is not inducing any autoreactive Th cells; and (iii) is generating strong non-self Th responses that help to produce high titers of anti-Aβ antibodies in wild-type and APPfTg mice(Agadjanyan et al., 2005; Mamikonyan et al., 2007; Petrushina et al., 2007). However, in these studies we used so called multiple antigenic peptide (MAP) backbone that is not easily can be scaled up for clinical studies and it is still required formulation in strong conventional adjuvant.

[001 1] In recent years, DNA vaccine technology has received a considerable scientific interest. DNA vaccines have some notable features in comparison with traditional vaccines, i.e., simplicity in production and storage, possibility in modification of genes encoding desired antigen/s, targeting the desired type of immune response (humoral or cellular). Intradermal administration of DNA vaccines using gene gun represents an efficient means of delivering DNA directly into dendritic cells (DC), the most potent professional antigen-presenting cells (APCs). After we reported for the first time on generation of the first DNA vaccine based on Ap₄₂ antigen(Ghochikyan et al., 2003), few other groups tested similar type of DNA vaccines in wild- type and transgenic animals(Kim et al., 2005; Kutzler et al., 2006; Okura et al., 2006; Qu et al., 2004; Schultz et al., 2004). However, in all these studies as well as in our experiments with APP/Tg mice (Cribbs and Agadjanyan, 2005) only low titers of anti-Aβ antibodies have been generated. It is alarming, because it is well known that DNA immunization works much better in mice than in large animals and humans(Babiuk et al., 2003; Gregersen, 2001). Thus, other vaccine design and delivery strategies, such as gen-gun immunization and eletroportaion should be considered for generation of potent humoral immunity in humans. Recently, such DNA epitope vaccine has been developed by our group and potency of this vaccine delivered by gene- gun or electropoartion has been demonstrated.

[0012] Another important obstacle for active vaccination of AD patients is hypo-responsiveness to vaccines in the elderly. Thus, immunosenescence in the elderly contributes to the poor outcome of vaccination and increased susceptibility of the elderly to infectious disease. Although all components of the immune system are affected in aged people, the immune response to vaccination is compromised mainly because of changes in adaptive immunity mediated by T cells. Vaccination can only be effective if antigen-specific T and B cells are present in the body (Nikolich-Zugich, 2005). These might be either naive CD4⁺T cells (T helper cells, Th) that require stimulation with a vaccine containing novel antigens, or memory Th cells that require expansion by previously encountered antigens. The Th cell population shifts to a lower ratio of naϊve to memory cells, and fewer naive Th cells are produced by the thymus with age. The involution of the thymus is almost complete at the age of 60 years and new Th cells can no longer be generated. In addition, the in vivo production of proinflammatory cytokines is upregulated in the elderly. In contrast to Th cells, no evidence for loss of B cell function has been found, although the B cell repertoire and the quality of antibody response may be affected by aging too. Brief Summary of the Invention

[0013] The present invention is directed to a method of treatment or prevention of Alzheimer's disease comprising administering of AD epitope vaccine to people with diagnosed early stage of AD or healthy middle-aged or older people with pre-existing memory Th cells generated by previous immunizations with conventional vaccines and/or induced after bacterial or viral infections (i.e. exposed to pathogens) in lifespan.

[0014] In one embodiment any portion of Aβ₄₂ can serve as the B cell epitope to induce antibodies binding to any form of Aβ (monomers, or oligomers, or fibrils). In another preferred embodiment, an irrelevant peptide (mimotope) can be used as the B cell epitope to induce antibodies to any form of Aβ.

[0015] In another preferred embodiment AB₄₂ or mimotope B cell epitope is presented in a plasmid in N>1 copies.

[0016] In another embodiment the T cell epitopes are any foreign natural T helper cell epitope (such as P2, P30, P32, P21, P23 form tetanus Toxin, epitopes from HbsAg or influenza) or synthetic promiscuous epitopes such as PADRE.

[0017] In one embodiment the molecular adjuvant is a cytokine (such as IL-4, IFNγ, GM-CSF, IL- 12, IL- 18), chemokine (such as MDC, βDF-3), complement component (C3d) or any other molecular adjuvant that could increase anti-Aβ antibody responses over the antigen that is not composed of a molecular adjuvant. In a preferred embodiment MDC is used.

[0018] In one embodiment the AD epitope vaccine is containing one, two, three or N copies of B cell epitope that can initiate production of anti-Aβ-antibodies when fused with foreign T cell epitope(s) or T cell epitope(s) and molecular adjuvant(s).

[0019] In another embodiment the AD epitope vaccine is the DNA epitope vaccine (see examples); recombinant protein epitope vaccine (see examples); peptide epitope vaccine (see examples); viral-like particles (VLP) epitope vaccine (such as HBV based epitope vaccine); viral replicons (VRP) epitope vaccine (such as alphavirus based VRP); viral vector epitope vaccine (such as adenovirus expressing AD epitope vaccine); or chimeric viruses (CV) expressing epitope vaccine (see examples).

[0020] In one embodiment the AD epitope vaccine is attached or encapsulated into nanoparticles. In another embodiment the recombinant protein or peptide epitope vaccine is attached to carrier protein

[0021] The present invention is also directed to a method of treatment or prevention of Alzheimer's disease comprising administering a AD epitope vaccine to a patient in need thereof.

[0022] The present invention is also directed to a method of treatment or prevention of Alzheimer's disease based on prime boost regimen (such as administering a DNA epitope vaccine to a patient in need thereof and boosting with recombinant protein vaccine, viral-like particles or viral vector encoding the epitope vaccine or any other heterologous vaccine combination).

[0023] The present invention is also directed to a method for generating a DNA vaccine or protein/peptide epitope vaccine comprising (a) construction and cloning of the minigenes into the mammalian expression vector (b) construction and cloning of the minigenes into the E.coli, yeast or baculovirus expression vector and purification of recombinant protein (c) immunizing an animal with a DNA or protein epitope vaccine (d) isolating sera from said animal that contains polyclonal antibodies generated in mice, APP/Tg mice, guinea pigs, dogs, rabbits, sheep, goats, chickens, etc as well as from Alzheimer's disease patients, (e) binding said polyclonal antibodies to Aβ4₂/4o/i5_>etc (f) measuring the avidity of binding of the said polyclonal antibodies to Aβ.

Brief Description of the Drawings

[0024] For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawing, in which:

[0025] Figure 1 show the expression of DNA epitope vaccines in CHO cells transiently transfected with the appropriate plasmid. Figure IA shows the expression of pMDC-Aβn- PADRE and pMDC-3Aβn-PADRE in the lysates of CHO cells by Western Blot (WB). Anti-Aβ 20.1 monoclonal antibody was used for staining. Figure 1 B shows the expression of p3Aβu- PADRE in CHO cells by IP/WB. 3Aβπ -PADRE protein was immunoprecipitated from lysate of CHO cells with Anti-Aβ 6E10 monoclonal antibody and analyzed by WB using 6E10 antibody for staining.

[0026] Figure 2 show CD4⁺ T cell response generated after gene gun immunization of mice with pMDC-Aβπ -PADRE and pMDC-3Aβn -PADRE. It shows that epitope vaccines induced proliferation of PADRE-specific but not Aβ-specific CD4⁺ T cells.

[0027] Figure 3 show that the vaccination of mice with pMDC-3Aβn -PADRE induced higher titers of anti-Aβ antibodies than vaccination with pMDC-Aβn -PADRE.

[0028] Figure 4 show the results of vaccination of mice by prime/boost regimen. Mice immunized twice with DNA epitope vaccine and boosted once with recombinant protein vaccine comprising the same immunogen generated significantly higher concentrations of anti-Aβ antibodies than mice immunized three times with DNA epitope vaccine.

[0029] Figure 5 show the role of molecular adjuvants in enhancing of immune responses. Figure 5 A shows that epitope vaccine attached to MDC induced significantly (PO.001) higher antibody response than epitope vaccine without adjuvant. Figure 5B shows that epitope vaccine attached to 3 copies of C3d component of complement induced significantly (PO.05) higher antibody response than epitope vaccine without adjuvant. Figure 5C shows that epitope vaccine attached to β-DF3 induced significantly (P<0.05) higher antibody response than epitope vaccine without adjuvant.

[0030] Figure 6 show that 2 copies of Aβ B cell epitope fused with T cell epitope in either orientation (N-terminus or C-terminus) equally potent in inducing of high titers of anti-Aβ antibodies.

[0031] Figure 7 show the results of immunization of mice with chimeric influenza virus expressing Aβ B cell epitope. Figure A shows the concentration of anti-Aβ antibody after immunization of mice with killed virus, or combination of live and killed virus. Figure B show the isotypes of anti-Aβ antibodies induced after immunization of mice with chimeric influenza virus.

[0032] Figure 8 show results of immunization of mice with protein-based epitope vaccine composed of one (MDC-Ap_n -PADRE) or three copies (MDC-3Aβn -PADRE) of B cell epitope. Figure 8A show that MDC-3Aβn-PADRE induced significantly higher antibody response than MDC-Aβn -PADRE. Figure 8B show that antibody generated after vaccination with MDC- 3Aβn-PADRE or 3Aβπ-PADRE bind to Aβ,j₂ peptide with higher avidity than antibody induced after MDC-Aβn-PADRE.

[0033] Figure 9 show results of the immunization of mice with or without pre-existing PADRE- specific memory T cells with DNA epitope vaccine. Mice possessing pre-existing memory T cells induced significantly higher antibody response than mice without pre-existing memory T cell.

[0034] Figure 10 show results of immunization of mice with DNA epitope vaccine encoding 3 copies of Aβ B cell epitope fused with 8 foreign T cell epitopes from conventional vaccines (Tetanus Toxin, HbsAg and influenza) fused with molecular adjuvant MDC. Figure 10 A show the proliferation of CD4⁺ T cells in splenocytes of immunized mice re-stimulated in vitro with each Th peptide, respectively. Figure 10 B show the percent of INFγ producing CD4⁺ T cells in splenocytes of immunized mice re-stimulated in vitro with each Th peptide, respectively. Figure 10 C show the percent of IL-4 producing CD4⁺ T cells in splenocytes of immunized mice re- stimulated in vitro with each Th peptide, respectively.

[0035] Figure 11 show the schematic representation of DNA-based epitope vaccine construct and its expression. Figure 10 A. show the construct, encoding MDC fused with 3 copies of self- Aβi-n epitope (Aβi.π) and multiple Th cell epitopes from conventional vaccines. Figure 11 B shows the expression of pMDC-3Aβ].π-Th construct in the lysate and supernatant of transfected CHO cells. Lane 1 represents the expression of pMDC-3Aβi-π-Th in the lysate. Lane 2 represents the expression of pMDC-3Aβi-n-Th in the supernatant. Lane 3 represents lysate of CHO cells transfected with control vector expressing irrelevant antigen gpl20 fused with MDC. Figure 11 C show the amino acid sequences of Th epitopes from conventional vaccines.

Detailed Description of the Invention

[0036] The Tg2576 mouse model (Hsiao et al., 1996) is one of the most widely used models of study amyloid deposition and expresses the Swedish APP mutation (APP 695.K670N-M671L) under the PrP mouse promoter, maintained on a hybrid C57BL/6xSJL background. These mice show a rapid increase in oligomeric Aβ levels at six months of age, and amyloid plaque deposition between nine and twelve months(Kawarabayashi et al., 2001; Lesne et al., 2006). In addition to intracellular Aβ, amyloid plaques, and oligomers, these mice recapitulate many of the neurological features of AD, including astrogliosis(Irizarry et al., 1997), microgliosis(Frautschy, 1998), cytokine production(Lim et al., 2000; Tan et al., 1999), oxidative stress(Pappolla et al., 1992; Smith et al., 1998), and dystrophic neurites(Irizarry et al., 1997). Tg2576 mice have significant cognitive impairment in spatial reference and working memory(Arendash et al., 2001a; Arendash et al., 2001b; Barnes et al., 1996; Hsiao et al., 1996; Kotilinek et al., 2002; Lesne et al., 2006; Morgan et al., 2000; Nicolle et al., 2003; Pedersen et al., 2006; Stackman et al., 2003; Westerman et al., 2002), the T-maze(Barnes et al., 2004; Corcoran et al., 2002), Y- maze(Ognibene et al., 2005), object recognition (Hale and Good, 2005; Ognibene et al., 2005) contextual fear conditioning(Corcoran et al., 2002; Quinn et al., 2007), and hippocampus- dependent trace conditioning(Ohno et al., 2006).

[0037] 3xTg-AD mouse model have the APPswe mutation, the tau (P301L) mutation, and are homozygous knockin for the PS-I (M146V) mutation(Oddo et al., 2003). 3XTg-AD mice typically exhibit features similar to AD pathology: (i) develop both Aβ plaque and neurofibrillary tangle (NFT) pathology in AD relevant brain regions, (ii) extracellular Aβ deposits prior to tangle formation; (iii) intracellular Aβ deposits; (iv) deficits in synaptic plasticity, including long-term potentiation (LTP) that occurs prior to extracellular Aβ deposition and tangles, but is associated with intracellular Aβ. Both strains of mice (APPTg2576 and 3XTg-AD) are very well characterized and are the best available AD animal-models representing AD-pathology. [0038] "Conventional vaccinations/vaccines" include, but are not limited to, Influenza, Hepatitis A, Hepatitis B, Tetanus, Diphtheria, Pertussis, measles, poliomyelitis, P. falciparum, rubella, etc.

[0039] "Natural infections" include, but are not limited to, influenza, Hepatitis A, Hepatitis B, Hepatitis C, Salmonella, Shigella, Listeria, Staphylococcus, Pneumococcus, etc.

[0040] "Memory T cells" are defined as CD4⁺T cells with a particular set of surface molecules providing a faster and stronger response of an animal after re-exposure to the same antigen.

[0041] "Pre-existing memory T cells" are memory T cells generated by previous immunizations with conventional vaccines and/or induced after bacterial or viral (pathogens) infections in lifespan.

[0042] "Diagnosis of AD" is conventionally made using cognitive and functional efficacy endpoints such as, but not limited to, Mini-Mental State examination (MMSE), neuropsychological test battery (NTB), Scale-Cognitive Subscale (ADAS-cog) and Disability Assessment for Dementia (DAD) {http://www.webmd.com/alzheimers/guide/making- diagnosis}. However, recently other molecular, biochemical, and other methods have been suggested and they are including, but not limited to, measuring tau/Aβ and ptau/Aβ ratio in the CSF (Fagan et al., 2007) and/or by screening for accumulation of Aβ in the brains using PIB- PET scan (Jack et al., 2008; Pike et al., 2007) or combination of Aβ₄₂ and PIB-PET scan {Fagan, personal communication} or any other agent instead of PIB and any other method that can be used for detection of amyloid depositions in the brain of living organism.

[0040] "Early stage of AD" is defined as the stage when a diagnostic marker such as CSF Aβ₄₂ level and antecedent biomarkers such as CSF tau/Aβ₄₂ and/or ptau/Aβ₄₂ predict future dementia in cognitively normal older adults(Fagan et al., 2007). Other early quantitative markers for AD pathology are 11 C-PIB-PET or 1 IC-PIB-PET combined with MIR. Strong relationship was shown between PIB binding and the severity of memory impairment in MCI, suggesting that individuals with increased cortical PIB binding are on the path to Alzheimer's disease(Jack et al., 2008; Pike et al., 2007). Also, other investigators have used other compounds, techniques, genetic markers, and biomarkers and combination of techniques and markers for early detection of AD.

[0041] "Before the diagnosis of AD" is defined are individuals who do not possess any diagnostic signs of AD, and can include individuals carrying genetic markers including, but not limited to, Familial Alzheimer's Disease (FAD) mutation, ApoE4 positivity, or Dawn syndrome (DS). This stage occurs before "early diagnosis of AD."

[0043] Aβ toxic forms include, but are not limited to, n-oligomers (dimers, trimers, tetramers, pentamer, hexamers....twelve-mers) protofibrils, ADDLs, fibrils, and even monomers(Cleary et al., 2005; Haass and Selkoe, 2007; Klyubin et al., 2005; Lesne et al., 2006).

[0044] "AD epitope vaccine" is the DNA epitope vaccine (see examples); recombinant protein epitope vaccine (see examples); peptide epitope vaccine (see examples); viral-like particles (VLP) epitope vaccine (such as HBV based epitope vaccine); viral replicons (VRP) epitope vaccine (such as alphavirus based VRP); viral vector epitope vaccine (such as adenovirus expressing AD epitope vaccine); or chimeric viruses (CV) expressing epitope vaccine (see examples) comprising one or more copies of B cell epitope, non-self Th cell epitope not possessing to Aβ₄₂ peptide and/or molecular adjuvant(s) that collectively induced anti-Aβ antibodies.

[0045] "DNA epitope vaccine" refers to a polynucleotide sequence that encodes a polypeptide comprising one or more copies of B cell epitope, T cell epitope not possessing to Aβ₄₂ peptide and/or molecular adjuvant that collectively induced anti-Aβ antibodies.

[0046] "A recombinant protein/peptide/polypeptide epitope vaccine" refers to a peptide/polypeptide amino acid sequence that expresses one or more copies of B cell epitope, T cell epitope not possessing to Aβ₄₂ peptide and/or molecular adjuvant that collectively induced anti-Aβ antibodies. [0047] "Peptide possessing any Aβ₄₂ B cell epitope but not self T cell epitope" are defined as the portion of Aβ that is immunogenic and may induce the production of antibodies capable of binding any form of Aβ₄₂ peptide such as monomeric, oligomeric, protofibrils and fibrils, but not able to induce the activation of T cells specific to Aβ₄₂.

[0048] "Any peptide possessing B cell epitope but not Aβ_42-self T cell epitope" are defined as the any peptide that could induce the production of antibodies capable to bind any form of Aβ₄₂ peptide such as monomeric, oligomeric, protofibrils and fibrils, however is not able to induce the activation of T cells specific to Aβ₄₂.

[0049] Recognition of B cell epitopes by immune cells of the individual causes changes in immune cell activity such as, but not limited to production of detectable titers of anti-Aβ antibodies in the sera of vaccinated people.

[0050] A B cell epitope - is a peptide that can (i) stimulate production of anti-Aβ antibodies that bind to monomeric and/or oligomeric, and/or fibrillar forms of Aβ and/or (ii) be recognized by B cell expressing immunoglobulin receptors (BCR) specific to any form of Aβ. For example, this B cell epitope can be, but not limited to, any part of Aβ₄₂ peptide or any irrelevant peptide that will mimic Aβ epitopes (i.e. mimotope).

[0051] Any part of Aβ₄₂ peptide may be used for B cell recognition as we demonstrated by binding of anti-Aβ antibodies to overlapping peptides. For instance peptides comprising 1-3, 2- 6, 1-7, 4-10 and 1-11 can be used.(Bard et al., 2003; McLaurin et al., 2002) The working examples demonstrate this using principle using Aβi.π or Aβuis Some divergence of these sequences not "conserved" will still result in functional activity and generation of anti- Aβ antbiodies. Such divergence can be at least 50%; preferably, at least 20%, and more preferably at least 10%, and more preferably 5%. Divergence can result from" sequence- conservative'Variants and "function-conservative variants". "Sequence-conservative variants" of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. "Function- conservative variants" are polynucleotide sequences that code for proteins wherein at least one given amino acid residue has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art.

[0052] Mimeotopes, which are polypeptides of unrelated sequence but with a 3-dimensional structure corresponding to the Aβ₄₂ peptide , that immunologically function in an identical. Mimeotopes, which are any biological molecule that is unrelated to Aβ₄₂ peptide structure, but has identical 3-d antigenic epitope/s and can be recognized by anti-Aβ B cells. An antibody for a given epitope antigen will recognize a mimotope which mimics that epitope. Vaccines utilizing mimotopes are being developed." Examples of mimotopes that can be used include, but are not limited to example (see below)

[0053] "Non-self T cell epitopes/foreign T cell epitopes" are any non-self peptide that can cause changes in immune cell activity such as, but not limited to production of cytokines, chemokines, and help B cells to produce detectable titers of anti-Aβ antibodies (recognized any form of this molecule) in the sera of vaccinated people. Examples of non-self T cell epitopes that can be used are, but not limited to PADRE, P2, P30, P32, P21, P23 form tetanus Toxin, epitopes from HbsAg, HBV nuclear capside or M protein of influenza. The DNA sequence of these non-self T cell epitopes are well known. Some divergence of from these sequences will still result in functional activity. Such divergence can be at least 50%; preferably, at least 20%, and more preferably at least 10%. Divergence can result in "sequence-conservative"variants and "function- conservative variants". "Sequence-conservative variants" of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. "Function-conservative variants" are polynucleotide sequences that code for proteins wherein at least one given amino acid residue has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art.

[0054] "Promiscuous T cell epitopes" are defined as any peptide that can be recognized by many DR molecules of the immune system and induce changes in immune cells of these individuals such as, but not limited to production of cytokines, chemokines, and help B cells to produce detectable titers of anti- Aβ antibodies in the sera of vaccinated people.

[0055] "Artificial T cell epitopes" can be used. These are defined as synthetic Promiscuous T cell epitopes included, but not limited to PADRE.

[0056] Molecular adjuvants are biological molecules that can modulate immune system in a such way that they will response to immunizations with any vaccine or antigen. A molecular adjuvant can bind to immune cells surface receptor directly or targeting moiety of other molecules that are ligand that binds to the receptor. It is particularly preferred that the cell surface receptor be an immunomodulatory receptor. Suitable cell surface receptors include, but are not limited to, CCR4 (for MDC), CCR6 (for β-DF3) IL-4R, IFNγR, CD21 (for C3d), and Toll-like receptors (TLR).

[0057] "Heterologous prime/boost vaccination" is defined as a vaccination regime using the combination of either DNA epitope vaccine, polypeptide/peptide/recombinant protein epitope vaccine (i.e. a polypeptide containing epitope recognized by B cells expressing BCR specific to Aβ, a non-self T cell epitope/s and molecular adjuvant/s), a virus expressing epitope recognized by B cells expressing BCR specific to Aβ only or in conjunction with a non-self T cell epitope/s and molecular adjuvant/s; or viral particles containing epitope recognized by B cells expressing BCR specific to Aβ only or in conjunction with a non-self T cell epitope/s and molecular adjuvant/s, or any other combination of heterologous vaccines.

[0058] Many different methods are used to facilitate uptake of DNA and peptide vaccines in animals. These include, but are not limited to using DC, nanoparticles, conventional adjuvant, patches, etc. [0059] When DNA is used as a vaccine, the DNA can be administered directly using techniques such as delivery by intramuscular or intradermal injection followed by electroporation, delivery on gold beads (gene gun), delivery by liposomes, or direct injection, among other methods known to people in the art. A preferred method of DNA epitope vaccination is including, but not limited to gene-gun and electroporation-based DNA delivery technologies. These methods involved in dramatic increase the efficiency of delivery of DNA into cells, and has demonstrated the capacity to increase the potency of DNA vaccines by -100 and more fold compared to conventional injection methods

[0060] Positive changes in neuropathology can be, but not limited to, reduction/inhibition of soluble and insoluble Aβ depositions in brain parenchyma and vasculature, less stimulation of glial activation, reduction change of tau in CNS (CFS and brains), inhibition/prevention of neuronal loss and degeneration of neurons, inhibition of cognitive decline and/or improvement of cognitive function.

[0061] Improvement in cognitive function can be measured by, but not limited to, using Scale- Cognitive Subscale (ADAS-cog) and Disability Assessment Scale for Dementia (DAD), Mini- Mental State examination (MMSE), neuropsychological test battery (NTB).

[0062] "Derivatives" include sequence-conservative variants and function-conservative variants. "Sequence-conservative variants" of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. "Function-conservative variants" are polynucleotide sequences that code for proteins wherein at least one given amino acid residue has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art.

[0063] "Vaccine formulation" of the present invention comprised an immunogenic amount of AD epitope vaccine with a pharmaceutically acceptable carrier. [0064] An "immunogenic amount" is an amount of AD epitope vaccine sufficient to evoke an immune response in the subject to which the vaccine is administered. The amount administered is an amount that induces a desired immune response, and the desired degree of protection against AD, inhibition or stabilization of AD pathology or therapeutic effect. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sterile pyrogen-free water and sterile pyrogen-free physiological saline solution.

[0065] The vaccine formulations of the present invention are suitable for patients diagnosed with early-stage of Alzheimer's disease. The vaccine formulations of the present invention are also suitable for patients known to have a genetic susceptibility to AD including but not limited to FAD mutation, positive for ApoE4 allele, patients with DS. In addition, the vaccine formulations of the present invention are suitable for the general population at large, including those without AD or without a genetic susceptibility to AD, who wish to invoke protection against contracting AD.

[0066] Administration of the vaccine formulation may be carried out by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), intradermal, intravenous, nasal, or to an airway surface. Topical application of the virus to an airway surface can be carried out by intranasal administration (e.g. by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally). Topical application of the virus to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the replicon as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed. An "immunogenic amount" is an amount of the replicon particles sufficient to evoke an immune response in the subject to which the vaccine is administered. Any vaccine can be use in any combination effective to elicit an immunogenic response in a subject. Generally, vaccine administered may be in an amount that will induce a desired immune response, and the degree of desired effect. Precise amounts of the vaccine to be administered may depend on data generated after clinical trials. [0067] The vaccine may be given in a single dose schedule, or preferably a multiple dose schedule that will induce a desired immune response, and the degree of desired effect. Precise schedule of the vaccine to be administered may depend on data generated in clinical trials.

[0068] By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a peptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the peptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polypeptide is at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in SEQ ID NO:2 can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 5371 1). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

[0069] It should also be noted that when referring here to the nucleic acid sequence is at least 90% identical to the each specific portions of the B cell epitope, non-self T cell epitope and molecular adjuvant sequences as found in a sequence selected from any of the sequences mentioned, this will result in nucleic acid sequence with an overall identity of less than 90%, because at least 90% identity refers to each portion of the entire construct. [0070] The present invention provides several approaches to overcoming the limitations of the prior art. One possible approach to overcome the limitations of the prior art is regular vaccination of healthy people or people with very early-stage AD, which can be diagnosed by measuring tau/Aβ and ptau/Aβ ratio in the CSF and/or by screening for accumulation of Aβ in the brains using PIB-PET scan and combination of these methods, and subsequently providing yearly booster vaccinations. Thus, early vaccination is important.

[0071] Another possible strategy to counteract the immunosenescence is to recruit previously generated memory Th cells produced during life time vaccination including childhood vaccination or during prior exposure to human pathogens. The majority of people already possess a broad panel of specific memory Th cells against conventional vaccines and different pathogens. Vaccination of these people with a recombinant vaccine composed of a B cell epitope of the desired antigen (inducing anti-Aβ antibody responses in case of AD vaccination) and non-self Th epitopes from conventional vaccines or pathogens described above, may boost the pre-existing memory T cells to expand rapidly and differentiate into effector Th cells, which leads to a faster and stronger antibody response specific to B cell epitope/s of any form of Aβ (Figure 12). Tetanus toxin (TT), influenza virus and HBV are examples of such conventional vaccines. As a result of mandatory vaccination against TT, yearly vaccination and/or infection with influenza, and vaccination against HBV, almost everyone should have memory CD4⁺T cells specific to helper epitopes in TT, HBV and conserved epitopes in influenza. Thus, a booster injection with AD epitope vaccine containing these conventional Th epitopes along with a strong molecular adjuvant should be enough for the rapid activation of pre-existing anti-TT, anti-HBV and anti-flu memory Th cells, resulting in the activation of B cells expressing anti-Aβ immunoglobulin receptors (BCR) and induction of strong and potent anti-Aβ antibody production in the elderly.

[0072] The invention provides AD epitope vaccines composed of several copies of self B cell epitope, foreign T cell epitope PADRE or multiple Th epitopes from Tetanus Toxoid, HBV and Influenza (such as P2, P30, P32, P21, P23, HbsAg, HBVnc, Influenza MT ) and molecular adjuvants (such as MDC, β-DF3, 3C3d). AD Epitope vaccine improve features of AD vaccine due to: (i) several copies of B cell epitope enhance the antibody response and avidity to the antigen; (ii) several copies of B cell epitope enhance antigen uptake and presentation of appropriate peptides by APC to T cells (iii) beneficial attachment of self B cell epitope to the promiscuous foreign T cell epitope can brake the tolerance and induce more robust immune response; replacement of self T cell epitope of Aβ₄₂ peptide with foreign epitope prevents the development of autoreactive T cell responses (iii) replacement of self T cell epitope with Th epitopes from conventional vaccines or pathogens to which human population is frequently exposed allow to boost pre-existing memory T cells generated during lifetime in the majority of population with AD epitope vaccine and induce rapid and robust anti-Aβ antibody response (iv) attachment to molecular adjuvant or formulation into molecular adjuvant significantly enhance and bias the immune response to the desired type (v) priming with DNA epitope vaccine and heterologous boost with protein/peptide, VLP or viral vector containing epitope vaccine allow to induce higher titers of ant-Aβ antibodies than that in case of homologous boost with DNA vaccine.

VACCINES

[0073] The present invention discloses the idea for the generation of AD epitope vaccine composed of several copies of B cell epitope fused with non-self promiscuous T cell epitopes from conventional vaccines or pathogens to which human population is frequently exposed and attached or mixed with molecular adjuvant to generate safe and potent anti-AD vaccine for immunization of people with diagnosed early-stage AD or healthy middle-age or elder people possessing pre-existing memory T cells. As example we are using constructed DNA epitope vaccine and purified protein vaccine for immunization of APP/Tg mice to analyze extent of pathology and behavioral impairment.

EXAMPLE 1

[0074] Immunization of APP/Tg mice with DNA epitope vaccine composed of one or three copies of B cell epitope of Aβ₄₂, non-self Th cell epitope, and a molecular adjuvant. [0075] Here, we engineered a plasmid vaccine in which a foreign Th cell epitope (PADRE) was fused with 1 or 3 copies of the B cell epitope of Aβ₄₂ (3Aβi.π). Additionally, in this plasmid we used macrophage-derived cytokine (MDC), a molecular adjuvant that, as we and others described previously, dramatically enhanced Th2 type of immune responses(Biragyn et al., 2001). Expression of resulted pMDC-Aβi.n-PADRE and pMDC-3Aβi_-π-PADRE and construct encoding epitope vaccine without molecular adjuvant (p3Aβ 11 -PADRE) has been confirmed in transiently transfected CHO cells by Western Blot (Figure 1). DNA epitope vaccines were tested in -3-4 mo-old 3xTg-AD mice. Three immunizations of 3xTg-AD mice with DNA epitope vaccine induced CD4⁺T cell response directed against foreign antigenic determinant, PADRE, but not against self-Aβ antigen (Figure 2). This cellular immune response was Th2-polarized, as analyzed by flow cytometry assay for detection of intracellular cytokines (IFNγ and IL-4). CD4⁺T lymphocytes provided B cells with sufficient help to produce high concentrations of anti- Aβi.π antibodies in vaccinated mice. Importantly the concentration of antibodies was significantly higher in mice immunized with DNA epitope vaccine containing 3 copies of B cell epitope than vaccines containing one copy (Figure 3). In addition the anti-Aβ polyclonal antibodies generated by MDC-3Aβi.n -PADRE had higher avidity to Aβ₄₂ peptide than antibodies generated by MDC-Aβi.n-PADRE. Thus, AD epitope vaccine is designed that is immunogenic in APP/Tg mice.

[0076] MDC molecular adjuvant was essential not only for generation the higher concentration of antibodies but also for directing the humoral immune response toward strict Th2-polarization (IgGl/IgG2c ratio for p3Aβi-n-PADRE is 2.2, and for pMDC-3Aβ_{M 1}-PADRE is 22.7). Collectively, the analysis of cellular and humoral immune responses demonstrated that the (i) vaccine containing several copies of B cell epitope induced production of higher concentrations Aβ-specific antibodies with higher avidity than vaccine containing one copy of epitope; (ii) DNA epitope vaccine did not activate autoreactive anti-Aβ T cells; (iii) Molecular adjuvant MDC additionally enhanced immune response and biased it toward anti-inflammatory Th2 type immunity that may be beneficial for elderly people.

[0077] DNA vaccination has rescued age-related cognitive decline in 3xTg-AD mice. [0078] 3xTg-AD mice vaccinated with DNA epitope vaccine demonstrated improvement in cognitive functions compared with age-matched naϊve transgenic animals. Our data demonstrated that vaccinated mice do not have difficulties in retaining information as non- vaccinated mice do, i.e. both short-term (1.5 hr) and long-term (24 hrs) spatial reference memory was rescued in immune animals. Antibody generated after AD epitope vaccine are potent.

[0079] Next, to analyze the mechanism elucidating the effect of DNA vaccination on behavioral improvement, we analyzed neuropathological changes in 18±0.5 mo old immune and control mice. First we tested Aβ depositions and demonstrated that vaccinations significantly reduced amyloid burden (diffuse and cored plaques) in the different areas of the brains of immune mice versus control groups. Epitope vaccine induced statistically significant reduction of both soluble and insoluble Ap₄₂ (PO.001) in the brains of immune mice. Insoluble Aβ₄o level was also decreased in vaccinated animals, but this decrease was not statistically significant. Vaccination with DNA epitope vaccine significantly decreased inflammation related pathology (microglial activation, astrocytosis) without increasing the incidence of cerebral microhemorrhages in aged 3xTg-AD mice. Tau pathology was also decrease in vaccinated mice compare it with that in control animals. No infiltration of T cells into the brains of mice immunized with epitope vaccine was observed(Movsesyan et al., 2008).

[0080] EXAMPLE 2

[0081] Prime/boost regimen were tested in 3xTg-AD mice, when mice were immunized twice with DNA epitope vaccine and boosted with the homologous recombinant protein. The concentration of antibodies was significantly higher in mice immunized with DNA epitope vaccine and boosted with protein than that in mice immunized with DNA vaccine only (Figure 4).

[0082] EXAMPLE 3

[0083] Attachment of epitope vaccine to molecular adjuvant enhances immune responses [0084] DNA epitope vaccine fused with molecular adjuvants MDC, β-DF3 or 3C3d induced significantly higher antibody responses in mice than DNA epitope vaccine lacking molecular adjuvant (Figure 5). Four groups of C57BL6 mice were immunized three times biweekly with pMDC-3Aβ_π -PADRE, pβDF3-3Aβ_π -PADRE, p3Aβ_π-PADRE-3C3d and p3Aβn-PADRE, respectively, using gene gun. Sera were collected on 10^th day after each immunization and anti- Aβ antibody concentration were measured by ELISA.

[0085] EXAMPLE 4

[0086] Peptide epitope vaccine._Two types of epitope vaccines based on peptide were generated: 2Aβi-n-PADRE and PADRE-2Aβi_-n attached to lysine backbone (2Aβ,.ii-P ADRE-MAP or PADRE-2Aβi.ii-MAP). Both peptides formulated in QuilA adjuvant induced equally high titers of anti-Aβ antibodies after three biweekly immunizations of C57BL6 mice (Figure 6). For further analysis of therapeutic potency of peptide-based epitope vaccine 9mo old APP/Tg 2576 mice possessing moderate AD-like pathology were immunized with 2Aβi.π -PADRE-MAP formulated in QuilA, and neuropathological changes in the brains of immunized mice were analyzed (Petrushina et al., 2007). Vaccination of APP/Tg 2576 mice with pre-existing AD-like pathology with epitope vaccine reduced insoluble Aβ deposition without any associated adverse events, such as CNS T cell or macrophage infiltration or microhemorrhages. However, vaccination did not alter the levels of the most toxic soluble oligomeric Aβ. These observations suggest that preventive vaccination and/or early therapeutic vaccination may protect from an AD, or may delay an onset of a disease, whereas therapeutic vaccination itself cannot disrupt the toxic oligomers, and may only minimally inhibit pre-existing AD pathology.

[0087] Viral vector based vaccine. As an alternative platform for delivery of immunogenic B cell epitope of Aβ₄₂ we recently developed and tested the recombinant influenza virus vectors that do not only deliver Aβi.io or any other peptide of interest to the host immune system, but also provide necessary T cell help. Recombinant influenza virus A/WSN/33 (HlNl) expressing B cell epitope of Aβ₄₂, Aβi.io (flu-Aβi.io) inserted into HA gene was constructed using plasmid- based reverse genetic rescue system(Schickli et al., 2001). C57BL/6 mice were immunized and boosted twice with different combinations of attenuated live or killed virus (Table 1).

Table 1. Monthly immunization of C57BU6 mice with combinations of live or killed recombinant influenza virus

[0088] Recombinant influenza virus expressing Aβi.io induced significant antibody response in all combinations, however a response was stronger when mice immunized 2 times with attenuated live virus were boosted with killed vaccine (Figure 7). All groups of mice immunized with recombinant virus induced predominantly antibodies of the IgG2b and IgG2a^b isotypes. The ratio of IgGl/IgG2a^b was less than 1, which implies that we induced predominantly Thl-type of immune response. This ThI response should not induce unwanted adverse events because it is specific to influenza virus, but not to self Aβ antigen.

[0089] Protein-based epitope vaccine. Protein-based epitope vaccines MDC-Aβn -PADRE and MDC-3Aβπ-PADRE induced robust antibody response after immunization of C57BL6 mice without any conventional adjuvant (Figure 8) however antibody response induced by vaccine containing three copies of B cell epitope is significantly higher than that induced by vaccine containing one copy of B cell epitope. For production of protein-based epitope vaccines MDC- 3Aβi_-π-PADRE and MDC-Aβ,_-π -PADRE minigenes were sub-cloned into pET24d E. coli expression vector in frame with a C-terminal His-Tag in order to produce recombinant protein in E. coli. Recombinant proteins were purified by Ni-NTA column from E. coli transformed with pET-MDC-3Aβi_-π -PADRE or pET-MDC-Aβ_Mi-PADRE followed by additional purification by Endotoxin removing gel. The purity of proteins was analyzed by 10% Tris-SDS-PA gel electrophoresis. Protein bands were visualized using Coomassie blue staining of the gel (Figure 8). Mice were immunized with 50μg protein subcutaneously four times biweekly and sera were collected on 10^th day after each immunization. [0090] EXAMPLE 5

[0091] Testing the potential of pre-existing memory T cells.

[0092] 1. Mice were immunized with DNA vaccine encoding PADRE T helper epitope fused with molecular adjuvant MDC. After 3 months of resting period, when PADRE-specific memory T cells were generated, mice were boosted with DNA epitope vaccine encoding MDC- 3Aβi.ii-PADRE minigenes. Control mice without pre-existing memory T cells were immunized once with DNA epitope vaccine and anti-Aβ antibody responses were compared in two groups. Mice with pre-existing memory T cells generated significantly higher concentration of anti-Aβ antibodies than mice without memory T cells (Figure 9).

[0093] 2. DNA epitope vaccine encoding 3 copies of Aβ₄₂ B cell epitope (3Aβπ) and 8 different T helper cell epitopes from conventional vaccines fused with MDC (pMDC-3Aβn-Th) were constructed and tested in C57BL6 mice (Figure 1 1). Two groups of mice were immunized with MDC-3Aβn-Th or control vector. Mice were bled and then sacrificed on the 7^th day after the last boost and cellular and humoral immune responses were analyzed. More specifically, we examined CD4⁺T cell proliferation and production of IL-4, IFNy cytokines (Figure 10) by FACS methods. Re-stimulation of splenocytes isolated from mice immunized with pMDC-3Aβn-Th with a mixture of all eight peptides induced strong T cell proliferation. Importantly, CD4⁺T cells from immune mice were activated vigorously with P32 or P21 peptides, moderately with P23 and HBV19-33, and weakly with INF17-31. pMDC-3Aβn-Th induced robust T cell proliferation specific to various Th epitopes from conventional vaccines, without activation of anti-Aβ T cell responses. Splenocytes isolated from mice injected with control vector did not proliferate after re-stimulation with any Th epitope or Aβ₄o peptide. Analyses of IFN γ (ThI- type cytokine) and IL-4 (Th2-type cytokine) by immune and control splenocytes support these data: (i) pMDC-3Aβn-Th activated production of both IFNγ and IL-4 by CD4⁺T cells after their in vitro re-stimulation with P32 and P21 and INFl 7-31, but not Ap₄₀ peptide (Figure 10). pMDC-3Aβn-Th induced robust humoral immune responses in mice (138.9 ± 68.9 μg/ml)

[0094] EXAMPLE 6 Amyloid fibrils could be recognized as foreign by the mature immune system since they are not present during its development. Using rabbit antisera raised against the oligomeric form OfAp₄₂, we have screened phage peptide library for the presence of foreign conformational mimotopes of Aβ. Antisera from both animals recognized predominately peptides with the EFRH motif from Aβ₄₂ sequence, however they recognized also several phage clones that mimic epitopes (mimotopes) within the fibrillar Aβ₄₂ but lack sequence homology with this peptide (Table 2).

Table 2. Detection of mimotopes 12 mer Phage Display Peptide Libraries and rabbit antisera generated against oligomeric form of Aβ₄₀

Sequence of Aβ₄₂ peptide: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGWIA

Mimotopes were synthesized, conjugated to KLH and used for immunization of rabbits. Sera were collected after the last immunization and antibodies recognizing Aβ₄₂ were measured by ELISA. One mimotope often induced antibodies (titer 1:5,000) recognized that Aβ₄₂ in ELISA as well as oligomeric and fibrillar Aβ₄₂ in dot blot assay.

[0095] Although the present invention has been described in connection with the preferred embodiments, it is to be understood that modifications and variations may be utilized without departing from the principles and scope of the invention, as those skilled in the art will readily understand. Accordingly, such modifications may be practiced within the scope of the following claims.

Extra information regarding DNA and aa sequences:

Nucleotide sequences

SEQ ID NO. 11 - MDC-3Ap,_-n-PADRE

1-279- MDC

280-309- spacer

310-342- first copy of Aβi.π

343-348 - spacer

349-381 - second copy of Aβi_n

382-387 - spacer

388-420 - third copy of Ap_1-, i

421-459 - PADRE

460-462 - stop codon

SEQ ID NO. 26 - MDC-3Aβi_-n-PADRE

1-93- MDC

94-103- spacer

104-114 -first copy of Aβi.n

115-116 -spacer

117-127 - second copy of Aβi.n

128-129 - spacer

130-140- third copy of Ap_{1 -π}

141-153 - PADRE

SEQ ID NO. 12- MDC-Aβi._n-PADRE

1-280- MDC 280-309- spacer 310-342 -AP_M i 343-381 - PADRE 382-384 - stop codon

SEQ ID NO. 27- MDC-Ap_1-π-PADRE

1-94- MDC 94-103- spacer 104-114 - Aβi.n 115-127 - PADRE

SEQ ID NO. 14- P-DF3-3Ap_Mi-PADRE

1-204- β-DF3

205-234-spacer

235-267- first copy of Ap_1-H

268-273 -spacer

274-306 -second copy of APi_-H

307-312 -spacer

313-345 -third copy of APi_-11

346-384 -PADRE

385-387 -stop codon

SEQ ID NO. 29- p-DF3-3Ap_1-π-PADRE

1-68- P-DF3

69-78 -spacer

79-89 -first copy of Ap, ., ,

90-91 -spacer

92-102 -second copy of Ap_1-11

103-104 -spacer

105-115 -third copy OfAp_1-11 1 16-128- PADRE

SEQ ID NO. 15- 3Ap,_-n-PADRE-C3d₃

1-78 signal sequence

79-111 first copy OfAp_1-H

112-117 spacer

1 18-150 second copy of Aβi-n

151-156 spacer

157- 189 third copy of Aβi.n

190-228 - PADRE

229-1125 - first copy of C3d

1126-1164- spacer

1165-2052 second copy of C3d

2053-2103- spacer

2104-2961- third copy of C3d

2962-2964 stop codon

SEQ ID NO. 30- 3Ap,_-n-PADRE-C3d₃

1-26 signal sequence

27-37 first copy of Aβ_{M 1}

38-40 spacer

41-51 second copy ofAβi.ii

52-53 spacer

54-64 third copy of Aβi.n

65-76 PADRE

77-375 first copy of C3d

376- 388 spacer

389-684 second copy of C3d

685-701 spacer 702-987 third copy of C3d

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Claims

The Claims

1. A composition comprising a nucleic acid sequence comprising a B cell epitope, a non-self T cell epitope and with a molecular adjuvant or without molecular adjuvant, wherein the nucleic acid encodes a polypeptide that initiates an immune response against Aβ in an individual.

2. The composition of claim 1, wherein said immune response is humoral or cellular.

3. The composition of claim 1, wherein said B cell epitope is any part of Aβ₄₂ peptide or a mimotope.

4. The composition of claim 3, wherein said B cell epitope is not a self T cell epitope.

5. The composition of claim 1, wherein said B cell epitope copy number is more than 1.

6. The composition according to claim 1, wherein said non-self T cell epitope is any foreign T cell epitope not possessing to Aβ₄₂ peptide.

7. The composition of claim 1, wherein said foreign T cell epitope is selected from the group consisting of P2, P30, P32, P21, P23 from Tetanus Toxin; epitopes of HbsAg; epitopes of influenza; and/or synthetic promiscuous epitopes.

8. The composition of claim 1, wherein said non-self T cell epitope is fused either at the N- terminus or at the C-terminus of the B cell epitope.

9. The composition of claim 1, wherein said molecular adjuvant is fused either at the N- terminus or C-terminus of the encoded polypeptide.

10. The composition of claim 1, wherein the nucleic acid sequence is selected from the group of SEQ. ID NO.l, SEQ. ID NO.2, and SEQ. ID NO.3 etc.,.

11. The composition of claim 1, wherein the nucleic acid sequence is at least 90% identical to the sequence selected from the group of SEQ. ID NO.l, SEQ. ID NO.2, and SEQ. ID NO.3 etc.

12. The composition of claim 1, wherein the nucleic acid sequence is at least 90% identical to the each specific portions of the B cell epitope, non-self T cell epitope and molecular adjuvant sequences as found in a sequence selected from the group of: SEQ. ID NO.l, SEQ. ID NO.2.. and SEQ. ID NO.3 etc.

13. A composition comprising a nucleic acid sequence comprising the B cell epitope and a non- self T cell epitope, wherein the nucleic acid encodes a polypeptide that initiates humoral immune response against Aβ in an individual.

14. The composition of claim 12, further comprising the addition of a nucleic acid sequence for a molecular adjuvant.

15. The composition of claim 13, wherein said molecular adjuvant is selected from the group consisting of MDC, β-DF3; the 3C3d, and IL-4 cytokine.

16. The composition of claim 13, wherein said molecular adjuvant sequence is selected from the group consisting of SEQ. ID NO.4 etc.

17. The composition of claim 1, further comprising a polypeptide containing a B cell epitope, a non-self T cell epitope and molecular adjuvant or without molecular adjuvant.

18. The composition of claim 15, wherein said polypeptide further contains a carrier protein that is either immunogenic or non-immunogenic.

19. The composition of claim 16, wherein said polypeptide is at least 80% identical to a sequence selected from the group consisting of SEQ. ID. No.5, SEQ. ID. No.6, SEQ. ID. No.7, etc.

20. An expression vector comprising the nucleic acid of claim 1 or nucleic acid encoding B and T cell epitope of claim 1 capable of inducing of anti-Aβ antibody responses.

21. A plasmid comprising the nucleic acid of claim 1.

22. A method for the generation of an immune response in an animal, including a human being, the method comprising presenting to the animal's immune system an immunogenically effective amount of a compound of claim 1, 16 or 17.

23. A method for the generation of an immune response in an animal, including a human being, the method comprising performing heterologous prime/boost regime to present to the animal's immune system an immunogenically effective amount of a compound selected from the group consisting of claim 1 ; a polypeptide containing a B cell epitope, a non-self T cell epitope and a molecular adjuvant; claim 19; claim 20; and claim 21.

24. The method of claim 23, wherein each member of said combination selected is presented either first or second.

25. The method of claim 22, wherein said animal is presented the compound before diagnosis of AD.

26. The method of claim 22, wherein said animal is presented the compound at early stage diagnosis of AD.

27. The method of claim 22, wherein said animal is presented the compound after diagnosis of AD.

29. A method for the targeting pre-existing memory Th cell populations of different specificity in individuals diagnosed, before diagnosis, or early diagnosis of AD comprising: presenting to a subject's immune system an immunogenically effective amount of a compound wherein said compound targets the pre-existing memory T cell population of the individual.

30. The method of claim 29, further comprising the step of immunomodulating the pre-existing memory T cell population to facilitate a humoral immune responses.

31. The method of claim 30, wherein the humoral immune response is production of antibodies specific to any form of Aβ (monomers, oligomers, fibrils).

32. The method of claim 29, wherein said pre-existing memory T cells were generated by prior exposure of the subject to conventional vaccines and pathogens.

33. The method of claim 29, 30, 31, 32, wherein compound is AD epitope vaccine which is DNA epitope vaccine containing the nucleic acid of claim 1.

34. The method of claim 29, 30, 31, 32, wherein compound is AD epitope vaccine which is VRP epitope vaccine containing the nucleic acid of claim 1 or nucleic acid encoding only B cell epitope capable of inducing of anti-Aβ antibody responses.

35. The method of claim 29, 30, 31, 32, wherein compound is AD epitope vaccine which is CV epitope vaccine containing the nucleic acid of claim 1 or nucleic acid encoding only B cell epitope capable of inducing of anti-Aβ antibody responses.

36. The method of claim 29, 30, 31, 32, wherein compound is AD epitope vaccine which is VLP epitope vaccine containing AD epitope vaccine of claim 17.

37. A method of treatment of Alzheimer's disease comprising administering of DNA epitope vaccine to a patient in need thereof using needle, gene gun, electroporation, Biojector, patches or any other delivery systems.

38. A method of treatment of Alzheimer's disease comprising administering of polypeptide delivered simultaneously and/or subsequently with any conventional adjuvant to a patient by any delivery system.