EA026787B1 - Nucleic acid construct and use thereof in treating, preventing and inhibiting alzheimer's disease - Google Patents

Abstract

The invention relates to a nucleic acid construct encoding a polypeptide that initiates an immune response against Aβ in a human. Provided is a method for the generation of an immune response in an animal and a human, and also a method of treatment of Alzheimer's disease comprising administering and effective amount of the claimed construct. Moreover, provided is a method for the generation of a cellular and humoral immune response in an animal and a human comprising performing preimmunization with the construct of the present invention and enhancing its action by administering the polypeptide encoded with said construct.

Description

(57) The invention relates to a nucleic acid construct encoding a polypeptide that induces an immune response to a human β-amyloid peptide. A method of inducing an immune response in animals and humans, as well as a method of treating Alzheimer's disease, comprising administering an effective amount of the claimed construct. In addition, a method is proposed for inducing a cellular and humoral immune response in animals and humans, including conducting primary immunization with the construct of the present invention and enhancing its effect by introducing a polypeptide encoded by the specified construct.

Cross references to related applications

The application for this patent claims the priority of provisional patent application US No. 60/966218, filed August 27, 2007, and provisional patent application US No. 61/124274, filed April 16, 2008.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for producing effective vaccines against Alzheimer's disease.

BACKGROUND OF THE INVENTION

Alzheimer's disease, characterized by a progressive decrease in cognitive functions, impaired memory and behavior, is the most common form of senile dementia. Alzheimer's disease is currently estimated to affect around 18 million people worldwide. It is estimated that by 2025 the number of patients will double and amount to 34 million people (1Sp: // \\ yo.ogd).

The neuropathological signs of this disease include neurofibrillary tangles (ΝΡΤ), deposition of β-amyloid (Av) in senile plaques, and loss of neurons in the affected areas of the brain (Pr. Aib §1voSha, 1994). These pathological changes lead to a significant loss of neurons and synapses in the course of the disease and, accordingly, increase the decrease in the patient’s functional capabilities. An important task in creating therapeutic approaches for the treatment of Alzheimer's disease is the identification of suitable targets. The Av peptide is cleaved from the amyloid precursor protein (APP) under the action of β and γ secretases (Ev1et apb U1Ge, 2001; §1, 1991; §1, 1994) and, apparently, plays the main role in the onset and development of Alzheimer's disease, on the basis of which the hypothesis of the amyloid cascade was put forward (Nagbu apb 8e1koe, 2002; Nagbu apb Ηί66ίηδ, 1992). It is known that there is an assumption that the most toxic forms of the Av ₄₂ peptide in the brain are soluble oligomers of various sizes (C1eagu e! A1., 2005; Sopde e! A1., 2003; K1uische! A1., 2005; Bee e1 a1 ., 2006; Evpe e! A1., 2006), currently there is no effective treatment for Alzheimer's disease and new preventive and / or therapeutic approaches for its treatment are needed. Recently, various approaches have been proposed for the treatment of Alzheimer's disease, aimed at reducing the level of Av in the brain or blocking the assembly of the peptide in pathological forms (Sopde e! A1., 2003; K1uish e! A1., 2005; Bogep / o apb Upppet, 1994 ), which violate cognitive functions (C1eagu e! a1., 2005), and among them was proposed immunotherapy.

A new and potentially effective approach to reducing the level of toxic forms of Av in the brain is immunotherapy, which, through Av-specific antibodies, can help eliminate amyloid deposits in the brain of APP transgenic mice (APP / TD) and patients suffering from Alzheimer's disease (Adab) apuap e! A1., 2005; Spbv apb Adab | apua1T 2005; §Syepk e! A1., 2004; Will leave apb Rtpke1, 2006). In these studies, it was shown that both passive (Wagb e! A1., 2000) and active (1api5 e! A1., 2000; Mozdap e! A1., 2000; §yepek e! A1., 1999) vaccination are extremely effective in mice and quite effective in humans (Rettet e! a1., 2004; Roh e! a1., 2005; Sbshap e! a1., 2005; Nosk e1 a1., 2003; Mavyay e! a1., 2005; No. so11 e! a1., 2003). However, the first immunotherapeutic clinical trial in patients with Alzheimer's disease, ΑΝ1792, was discontinued when some patients immunized with a vaccine containing Av ₄₂ and Ts821 experienced side effects (meningoencephalitis) in the central nervous system (Waueg e! A1., 2005; Reggeg e! A1., 2004; Roh e! A1., 2005; Obshap e! A1., 2005; Nosk e! A1., 2003; Mavyay e! A1., 2005; No.co11 e! A1., 2006; No. so11 e! a1., 2003; Ogdodo / oe! a1., 2003; Rayop e! a1., 2006; § Syepk, 2002; Will leave apb 8e1koe, 2002). Despite the fact that the true cause of the side effects remains unknown, all assumptions relate to autoreactive T cells specific for the T cell epitopes of Av, adjuvant (Ts821) and the use of the vaccine composition in phase 11a of the trials (§ Syepk, 2002; Ubet 8e1koe, 2002).

A low percentage of respondents (~ 19%) and generally low antibody titers in elderly patients in response to their own antigen (~ 1: 2200) even in the presence of a strong adjuvant (Waueg e! A1., 2005; Oishap e! A1., 2005; Ra !! op e! A1., 2006) are a significant obstacle to the application of the active immunization approach in elderly patients with a physiologically weakened immune system (Otekeskoebp8! Esp ap Huyuk 2002).

A recent six-year follow-up study of patients in the clinical trial group ΑΝ1792 showed that antibodies to Av can help resorb plaques in the brain of patients with Alzheimer's disease. The degree of resorption of the plaques varies greatly, but to some extent it correlates with the titers of antibodies to Av in the serum of vaccinated patients with Alzheimer's disease. It is important that the presence of plaques is not always a prerequisite for a progressive decrease in cognitive function in patients with Alzheimer's disease. The authors suggested that:

(ί) the presence of Av plaques is necessary for triggering, but not for the progression of neurodegeneration processes in patients with Alzheimer's disease;

(o) amyloid plaques are a secondary pathological phenomenon and extracellular

- 1 026787 soluble oligomeric or intra-neural forms of Αβ are responsible for neurodegeneration in Alzheimer's disease;

(ίίί) immunization is more effective for prevention than for treatment of the disease, since it can block the deposition of oligomeric forms of Αβ (Ho1tek e! a1., 2008).

Other approaches have been proposed as an attempt to overcome the difficulties associated with active immunization of elderly patients with Alzheimer's disease. One of them is a passive immunization strategy based on antibodies to Αβ. Recently, Elap Sogrogaiop presented detailed results of clinical trials lasting 18 months (phase 2), bapineuzumab (AAB-001) in patients with mild or moderate severity of Alzheimer's disease (1CO, 2008, Chicago, 1b). Bapineuzumab is the first humanized monoclonal antibody designed to eliminate toxic Av from the human brain. In order to evaluate the safety and tolerance to bapineuzumab in patients with mild to moderate Alzheimer's disease and to investigate the effectiveness of the dose level, a double-blind clinical trial plan was developed using increasing doses of the drug and placebo as a control. Statistically significant differences from baseline data were observed at 78 weeks in patients who were not carriers of ApoE4 who received bapineuzumab, both in relation to cognitive and functional disorders. Patients carrying ApoE4 showed no statistically significant differences for either cognitive or functional disorders. Side effects (PE) were usually mild to moderate, and the vasogenic edema observed in some patients appeared to be dose-dependent. It should be noted that clinical trial ΑΝ1792 showed the progression of cerebral amyloid angiopathy (CAA), and this coincides with the data that passive immunotherapy can also lead to increased CAA in transgenic mice and increase the incidence of microbleeding in vaccinated patients with Alzheimer's disease (RGegGeg e! A1., 2002; Kaske e! A1., 2005; KhUPsosk e! A1., 2004). All these data indicate that treatment should be started in advance (before the accumulation of toxic forms of amyloid in the vascular network).

Another approach used to develop a safe vaccine for the treatment of Alzheimer's disease is to try to avoid the T-cell response specific for its own antigen, Av. An example is the creation of an epitope vaccine consisting of small B-cell epitopes of Av ₄₂ and unrelated T helper (T) epitopes. E1ai apy HUUEY! have developed and are currently testing a conjugate vaccine (ACC-001, phase 2), in which small Ab terminal peptide fragments of Ab are conjugated to a carrier protein isolated from a non-toxic mutant diphtheria toxin and combined with T1 type adjuvant 0521 (No.№.е1аи .sot; 1Wr: // \ y \ y \ y.a1 / Gogit.ogd / ne \ y / ye! ap.akr'.ky = 1807).

A possible difficulty for the use of immunotherapy may be the fact that a decrease in the insoluble form of Av can lead to an increase in the level of soluble forms of this peptide (Ho1tek e! A1., 2008; Rayop e! A1., 2006), mainly oligomers that are most toxic to neurons and cause cognitive impairment (Naakk ap 5e1koe, 2007). Our recent preclinical studies of ίη ugo have shown that sub-stoichiometric concentrations of purified antibodies to Av prevent aggregation of Av ₄₂ and cause decomposition of the formed fibrillar forms of Av ₄₂ to non-toxic forms that do not have a fibrillar structure. However, antibodies to Av cannot destroy oligomers, although they reduce the formation of oligomeric forms of Av ₄₂ (Matkopuap e! A1., 2007). These observations, obtained by ίη ugo, suggest that therapeutic vaccination will not destroy the toxic oligomers ίη νί \ Ό. and it can only minimize the already existing manifestations of Alzheimer's disease, therefore, immunotherapy must be started before irreversible processes are caused by the deposition of Av peptide in the vascular network and brain parenchyma. In other words, if the development of pathology in Alzheimer's disease is associated with the accumulation of oligomeric forms of Av ₄₂ in the brain, then therapeutic vaccination will not be effective. Moreover, on the contrary, protective vaccination can be used to effectively prevent the development of Alzheimer's disease.

Recently, we published data on the effectiveness of the epitope peptide vaccine consisting of the immunodominant B-cell epitope of Av ₄₂ (Av _{4-1 5} or Av _1-11 ) together with a strong foreign T-cell epitope. The data obtained indicate that such a vaccine can be safe and immunogenic for humans, since it (ί) is highly affective for 15 of the 16 most common human HLA-EC molecules (A1exapieg e! A1., 2000; A1exapieg e! A1. , 1998; La Coca 2002; Rashpa-VogShdpop e! A1., 1989; XUeeg e! A1., 1999; XUe1 e! A1., 2001); (ίί) does not induce any autoreactive T cells; and (ίίί) elicits a strong non-autoimmune Tb response, which promotes the production of a high titer of antibodies to Av in wild-type and transgenic APP / TD mice (Aday. (Apuap e! A1., 2005; Matcopuap e! A1., 2007; Reikypa e! A1., 2007) However, in these studies, the inventors used the so-called multiple antigenic peptide (MAP) of the spine, which is difficult to obtain at the scale of clinical trials, so there remains a need for a composition with a strong standard adjuvant.

In recent years, DNA vaccine technology has been of significant scientific interest.

- 2 026787

Compared to traditional vaccines, DNA vaccines have important features, for example, such as ease of preparation and storage, the ability to modify genes encoding antigens of interest, and orientation towards the type of immune response of interest (humoral or cellular). Intradermal administration of DNA vaccines using gene cannons is an effective way to deliver DNA directly into dendritic cells (MS), the most powerful antigen presenting cells (APCs). After the authors of the invention first reported the receipt of the first DNA vaccine based on Αβ .- ^ antigen (SyosNkuap s1 a1., 2003), several other research groups also analyzed DNA vaccines of the same type in transgenic animals and wild animals type (K1sh s1 a1., 2005; Kp1 / 1sg s1 a1., 2006; Okiga s1 a1., 2006; Op s1 a1., 2004; 5> s1sh11 / s1 a1., 2004). However, in all these studies, as well as in our studies conducted on LRR / Td mice (Cn3b apf Adafapuap, 2005), only low titers of antibodies to ββ were produced. This caused some concern, as it is well known that DNA immunization is more effective in mice than in large animals and humans (BaSlk s1 a1., 2003; SGSBSGSP. 2001). Thus, to create strong humoral immunity in humans, it is necessary to use a different composition of vaccines and other methods of their delivery, for example, immunization with gene guns and electroportation. Recently, we managed to develop such epitope DNA vaccines, and the possibility of their delivery using gene guns or electroporation was shown.

Another important issue associated with active vaccination of patients with Alzheimer's disease is vaccine hyperactivity in the elderly. Thus, physiological weakening of the immune system contributes to poor vaccination efficacy and increases the susceptibility of older people to infectious diseases. Although all components of the immune system undergo changes in the elderly, a weak immune response to vaccination is associated mainly with changes in adaptive immunity due to T cells. Vaccination can only be effective if antigen-specific Ti B cells are present in the body (HocCon-2id1cb, 2005). This can be either naive SE4 'T cells (T-helpers, T1), which need stimulation with a vaccine containing new antigens, or T1 memory cells, the number of which should be large as a result of antigens previously acting on the body. With age, the population of T1 cells changes to a low ratio of naive cells versus memory cells, and the thymus produces an ever smaller number of naive T1 cells. By the age of 60, an almost complete regression of the thymus occurs and new T1 cells are no longer produced. In addition, the process of production of pro-inflammatory cytokines is activated in the elderly людейη νίνο. Unlike Ty cells, there is no evidence of loss of function by B cells, although the composition of B cells and the quality of the humoral response also change with age.

Summary of invention

The present invention relates to a nucleic acid construct encoding a polypeptide that induces an immune response to human β-amyloid peptide (Αβ) and contains a B-cell epitope that includes one or three copies of the Αβ _1-11 fragment, a T-cell epitope, which is alien to humans T-helper epitope RAOKE and molecular adjuvant, the nucleotide sequence of the construct is at least 90% homologous to the sequence selected from the group consisting of 8EC GO N0: 11, 12,14 and 15, and aminokis otnaya polypeptide sequence of at least 90% homologous to a sequence selected from the group consisting of DPP 8ETS N0: 26, 27, 29 and 30.

In one embodiment, said immune response is a humoral or cellular immune response. In yet another embodiment, said foreign Tcellular epitope is connected to the C-terminus of a B-cell epitope. In one embodiment, said molecular adjuvant is coupled to the Ν-terminus or to the C-terminus of the encoded polypeptide. In a particularly preferred embodiment, the sequence of the construct is selected from the group consisting of 8 ETS GO N0: 11, 12, 14 and 15 and the sequence of the construct is at least 90% homologous to the nucleic acid encoding the polypeptide. The molecular adjuvant may be selected from the group consisting of MES, β-0Ρ3, 3C3b.

The present invention also relates to an expression vector containing the indicated nucleic acid construct, which is capable of inducing a humoral response to Αβ, as well as to a plasmid containing the specified nucleic acid construct.

The present invention relates to a method for inducing an immune response in animals and humans, comprising presenting to the animal and human immune systems an immunogenic effective amount of said nucleic acid construct. In one embodiment, said animal and human receive said structure until a diagnosis of Olzheimer's disease is established. In another embodiment, the specified animal and man receives the specified design at an early stage of Olzheimer's disease or after the diagnosis of Olzheimer's disease.

The present invention also relates to a method for inducing an immune response in animals and humans, comprising administering a primary immunization with said construct and subsequent

- 3 026787 enhancing its action by introducing a polypeptide encoded by the specified design.

The invention relates to a method for targeted exposure of existing populations of Thelper cells of various specificity in patients diagnosed with Alzheimer's disease at an early stage, or until a diagnosis of Alzheimer's disease is established, including presenting the individual's immune system with an effective amount of the indicated nucleic acid construct. In one embodiment, such a method further comprises the step of immunomodulating an existing population of memory T cells to develop a humoral immune response. The indicated humoral immune response is the production of antibodies specific for any form of Av (monomeric, oligomeric, fibrillar), and the indicated existing memory T cells have arisen as a result of preliminary exposure of the corresponding vaccines and pathogens to the body. In one embodiment, the nucleic acid construct is an epitope DNA vaccine.

The invention also relates to a method for treating Alzheimer's disease, comprising administering to the patient an effective amount of said nucleic acid construct using a needle, a gene gun, electroporation, a pneumatic syringe Vu) ec1og, plasters and other delivery devices.

The invention also relates to a method for treating Alzheimer's disease, comprising administering to the patient an effective amount of said construct simultaneously and / or sequentially with any suitable adjuvant using any delivery method.

Brief Description of the Figures

For a more complete understanding of the present invention, the following is a description of the figures attached to the present description.

In FIG. 1 shows the expression of epitope DNA vaccines in CHO cells transfected with the corresponding plasmid. In FIG. 1A shows the expression of rMOS-Av ₁₁ -PAOKE and rMOS-3Av ₁₁ -PAOK in the CHO cell lysate, as determined by Western Blot (HC). For staining, a monoclonal antibody to Av 20.1 was used. In FIG. 1B shows the expression of p3Ab ₁₁ -PAOKE in CHO cells, determined by the method [P / ^ HC. Protein 3Av ₁₁ -PAOKE was isolated from CHO cell lysate by immunoprecipitation and analyzed by the HC method using 6E10 antibody for staining.

In FIG. Figure 2 shows the SI4 + T-cell response induced after immunization with rOSAb _11- RAOKE and rIIS-Zavp-RANKE mice using a gene gun. Epitope vaccines have been shown to induce proliferation of RAOKE-specific, but not Av-specific, SI4 + T cells.

In FIG. Figure 3 shows that vaccination of mice with rMIS-Zavp-RANKE induces a higher titer of antibodies to Av than vaccination with rMOS-Avp-RAOPE.

In FIG. 4 shows the results of vaccination of mice in the framework of rht-bob immunization. Mice twice immunized with an epitope DNA vaccine and a single recombinant protein vaccine containing the same immunogen produced significantly higher amounts of antibodies to Av than mice immunized three times with an epitope DNA vaccine.

In FIG. 5 shows the role of molecular adjuvants in enhancing the immune response. In FIG. 5A shows that an epitope vaccine coupled to MOC induces a significantly more pronounced humoral immune response (P <0.001) than an epitope vaccine without adjuvant. In FIG. 5B shows that an epitope vaccine combined with three copies of the C3b complement component induces a significantly more pronounced humoral immune response (P <0.05) than an epitope vaccine without adjuvant. In FIG. 5C shows that an epitope vaccine combined with bOP-3 induces a significantly more pronounced immune response (P <0.05) than an epitope vaccine without adjuvant.

In FIG. Figure 6 shows that 2 copies of the B-cell epitope of Av connected to the T-cell epitope in any direction (Ν-terminal or C-terminal) equally produce high titers of antibodies to Av.

In FIG. 7 shows the results of immunization of mice with a chimeric influenza virus expressing Av B cell epitope. In FIG. 7A shows the concentration of antibodies to Av after immunization of mice with a killed virus, or a combination of live and killed viruses. In FIG. 7B shows isotypes of antibodies to Av induced after immunization of mice with a chimeric influenza virus or a combination of live and killed viruses.

In FIG. Figure 8 shows the results of immunization of mice with a protein epitope vaccine consisting of one B-cell epitope (MOS-Avp-RAOKE) or three copies of the epitope (MOS-3Av _11- RAOKE). In FIG. 8A shows that MOS-3Av ₁₁ -PAOXE induces a significantly more pronounced humoral immune response than MOS-Avp-RAOKE. In FIG. 8B shows that antibodies produced after vaccination with MOS-3Av _11- RAOKE or 3Av _11- RAOKE bind to Av ₄₂ peptide with higher avidity than antibodies induced after administration of MOS-Avp-RANKE.

In FIG. Figure 9 shows the results of immunization with an epitope DNA vaccine in mice with or without RAOKE-specific memory T cells. In mice with preexisting memory T cells, the immune response was much higher than in mice without preexisting T cells

- 4,026,787 memory.

In FIG. 10 shows the results of immunization of mice with an epitope DNA vaccine consisting of 3 copies of an Άβ B-cell epitope connected to 8 foreign T-cell epitopes of known vaccines (tetanus toxin, HbAcD and influenza virus), and with molecular adjuvant MES. In FIG. 10Ά shows the proliferation of CE4 'T cells in splenocytes of immunized mice restimulated with ίη νίίΓΟ with each T cell peptide, respectively. In FIG. 10B shows the percentage of ΙΝΕγ-producing CE4 'T cells in splenocytes of immunized mice restimulated with ίη νίίΓΟ by each T-cell peptide, respectively. In FIG. 10C shows the percentage of 1L-4-producing CE4 'T cells in splenocytes of immunized mice restimulated with ίη νίίτο with each T cell peptide, respectively.

In FIG. 11 is a schematic representation of the construction of an epitope DNA vaccine and its expression. In FIG. 11Ά shows a construct encoding an MES coupled to 3 copies of the Αβ _1-11 epitope (Αβι _- ιι) and a plurality of T cell epitopes obtained from known vaccines. In FIG. 11B shows the expression of the pMES-ZAVM ^ Tb construct in the lysate and supernatant of transfected CHO cells. Lane 1 corresponds to the expression of pMES-ZAVM ^ Tb in the lysate. Lane 2 corresponds to the expression of rMES-ZAV ^ n-TB in the supernatant. Lane 3 corresponds to the lysate of CHO cells transfected with a control vector expressing the foreign antigen dr120 connected to the MES. In FIG. 11C shows the amino acid sequences of TB cell epitopes obtained from known vaccines.

DETAILED DESCRIPTION OF THE INVENTION

The mouse model Тd2576 (Н81ao οί а1., 1996) is one of the most widely used models for studying the process of amyloid deposition and expression of the Swedish APP mutation (ΆΡΡ695.Κ670Ν-Μ671Ε) under the control of the mouse Prg promoter, which supports von C75Bb / 6x§ hybrids 4b. These mice at the age of six months showed a rapid increase in the level of oligomeric forms of ββ, and amyloid plaque deposition occurred between nine and twelve months of age (KatagaLauase βί a1., 2001; Εβδηβ βί a1., 2006). In addition to intracellular Άβ, amyloid plaques, and oligomers, these mice show multiple neurological signs of Alzheimer's disease, including astrogliosis (Ιπ / аг гу βί a1., 1997), microgliosis (гагаШШззс, 1998), and the production of cytokines (ίίιιι βί а1, 2000. βί a1., 1999), oxidative stress (Rarro11a βί a1., 1992; διηίΐΐι βί a1., 1998) and dystrophy of neurons (Ιπ / argu βί a1., 1997). The Td2576 mice have significant cognitive impairment associated with spatial orientation and working memory (AgspbШ βί a1., 2001a; Agepbak βί a1., 2001b; Wag ^ z βί a1., 1996; H81ao βί a1., 1996; 81οίί1ίηβΕ βί a1. , 2002; Εβδηβ βί a1., 2006; Mozhdap βί a1., 2000; ΝΚο11β βί a1., 2003; ΡβάβΓδβη βί a1., 2006; §ίasktaη βί a1., 2003; ^ βδίβ ^ тaη βί a1., 2002), problems with behavior in the T-shaped maze (Wat ^ βί a1., 2004; Sogsogam βί a1., 2002), the Υ-shaped labyrinth (Θ ^ ηΛβηβ βί a1., 2005), object recognition (Nak apy Ooof 2005; Θ§ ηΛβηβ βί a1., 2005), by the development of conditioned reflexes of fading (Sogsoschp βί a1., 2 0 02; Οιιίηη βί a1., 2007) and conditioned reflexes to a trace stimulus, depending on the functioning of the hippocampus (GLPo βί a1., 2006).

3xTg-AE mice carry the mutation ΛΡΡδ ^ β, the mutation 1ai (P301b) and have a homozygous knockout for the mutation Pg-1 (M146U) (Oyo βί a1., 2003). 3xT§-AE mice usually have symptoms similar to those of Alzheimer's disease: (ί) they exhibit both Άβ plaques and neurofibrillary tangles (No. T) in the brain region similar to those of Olzheimer's disease, (ίί) extracellular deposition of Άβ occurs before the formation of tangles, (ίίί) Αβ is deposited inside the cells, (ίν) there is an increasing deficit in synaptic plasticity, including prolonged potentiation (LTP), which occurs before extracellular deposition of Άβ and the formation of tangles, but associated with intracellular accumulation of Άβ. Both mouse lines (APPTd2576 and 3xT§-AE) are very well described and are best suited as a mouse model in which the pathology of Olzheimer's disease is manifested.

The term “known vaccination / vaccines” means, but is not limited to, vaccines against influenza, hepatitis Ά, hepatitis B, tetanus, diphtheria, pertussis, measles, polio, R.TaUragit, rubella, etc.

The term natural infection is understood, but not limited to, infection with influenza viruses, hepatitis Ά, hepatitis B, hepatitis C; salmonella, shigella, listeria, staphylococcus, pneumococcus, etc.

The term memory T-cells is understood to mean CE4 'T cells with a specific set of molecules on the surface that provide a quick and strong response in an animal that was previously exposed to the same antigen.

The term “pre-existing memory T cells” refers to memory T cells resulting from an earlier immunization with known vaccines and / or induced by a bacterial or viral infection (pathogen action) during life.

The diagnosis of Alzheimer's disease is usually made on the basis of an assessment of cognitive and functional characteristics, using, for example, but not limited to, the method Μίηί-ΜβηΙη1 δίηίβ Exat ^ ηаί ^ οη MM8E (a brief scale for assessing the mental state), a set of neuropsychological tests (ΝΕΒ),

- 5,026,787 a scale for assessing cognitive function (ΑΌΑδ-sod) and a scale for assessing disability (ΌΆΌ) (1Wp: // xx \ x \ x. ELt.Sot / a1 / e1te5 / dIPe / so1pd - ShadpoX). Recently, however, molecular, biochemical, and other methods have been proposed for the diagnosis of Alzheimer's disease, including, but not limited to, measuring the ratios Ιαιι / Αβ and ρΙαυ / Λβ in cerebrospinal fluid (C8P) (Radap e! A1., 2007) and / or screening for Αβ deposition in the brain using an ΡΙΒ-сканирования scan (Lask e! a1., 2008; Ρίΐ / e e! a1., 2007) or a combination of Αβ42 and ΡΙΒ-ΡΕΤ scanning (Radap, personal message), or using any means other than ΡΙΒ and any other method that can be used to detect deposits a iloida in the brain of a living organism.

The term "early / early stage of Alzheimer's disease" refers to the stage at which the level of diagnostic markers, such as Αβ42 in C8P, and previously used biomarkers, such as Ι; ηι / Λβ ₄₂ and p! Ai / Av ₄₂ in C8P predict the occurrence of senile dementia in the future of a cognitively normal elderly person (Radap e! a1., 2007). Other early quantitative markers of Alzheimer's disease are PS-ΡΙΒ-ΡΕΤ and PS-ΡΙΒ-ΡΕΤ in combination with ΜΙΚ. A strong association has been shown between ΡΙΒ binding and the severity of memory impairment in mild cognitive impairment (MC1). which suggests a risk of developing Alzheimer's disease in patients with an increased cortical level of binding ΡΙΒ (Lask e! a1., 2008; Ρίΐ / e e! a1., 2007). In addition, other compounds, methods, genetic markers, biomarkers, and combinations of methods and markers were used in other studies for early diagnosis of Alzheimer's disease.

The term pre-diagnosis Alzheimer's disease is understood to mean individuals who do not show any diagnostic signs of Alzheimer's disease, and include individuals with genetic markers, including, but not limited to, mutations of the familial form of Alzheimer's disease (RAO), a positive reaction to ApoE4, or Down syndrome . This stage appears before the initial / early stage of Alzheimer's disease.

Toxic forms of Av include, but are not limited to, p-oligomeric (dimeric, trimeric, tetrameric, pentameric, hexameric-twelve-dimensional) protofibrils, ΑΌΌΕ, fibrils, and even monomers (C1eagu e! A1., 2005; Naa88 apb §1koe, 2007; К1уишш!! A1., 2005; Lekpe e! A1., 2006).

The epitope vaccine against Alzheimer's disease is an epitope DNA vaccine (see examples); recombinant protein epitope vaccine (see examples); peptide epitope vaccine (see examples); virus-like particle epitope vaccine (e.g. ΗΒν-based epitope vaccine); viral replicon epitope vaccine (e.g., alphavirus replicon epitope vaccine); an epitope vaccine based on viral vectors (for example, an epitope vaccine based on an adenoviral vector); or an epitope vaccine created on the basis of chimeric viruses (see examples) containing one or more copies of extracellular epitopes, foreign Τΐι-cell epitopes absent in the Λβ42 peptide, and / or molecular adjuvant (s), which together induce the production of antibodies to Av .

The term epitope DNA vaccine is understood to mean a polynucleotide sequence that encodes a polypeptide containing one or more copies of B-cell epitopes, T-cell epitopes that are absent in the Av42 peptide, and / or a molecular adjuvant that together induce the production of antibodies to Av.

The term recombinant protein / peptide / polypeptide / epitope vaccine refers to a peptide / polypeptide amino acid sequence that represents one or more copies of B-cell epitopes, T-cell epitopes that are absent in the Av ₄₂ peptide / and / or molecular adjuvant that together induce production antibodies to Av.

The term peptide containing any B-cell epitope of Av ₄₂ , but lacking an epitope for its own T cells, means the part of Av that is immunogenic and can induce the production of antibodies capable of binding to any form of the Av ₄₂ peptide, including monomeric and oligomeric forms , protofibrils and fibrils, but is not able to induce the activation of T cells specific for Av ₄₂ .

The term peptide containing a B-cell epitope but lacking the Av ₄₂ epitope for its own T cells means any peptide that can induce the production of antibodies capable of binding any form of the Av ₄₂ peptide, including monomeric and oligomeric forms, protofibrils and fibrils, but not able to induce activation of T cells specific for Av ₄₂ .

Recognition of B-cell epitopes by the immune system of individuals leads to a change in the activity of cells of the immune system, for example, but not limited to, the production of detectable antibody antibody ranges in serum of vaccinated people.

A B-cell epitope is a peptide that: (ί) can stimulate the production of antibodies to Av, which can bind monomeric and / or oligomeric and / or fibrillar forms of Av; and / or (ίί) is recognized by expressed B-cell immunoglobulin receptors (ΒΟΚ.) specific for any form of Av. A B-cell epitope can be, for example, any part of the Ab peptide or any foreign peptide that will mimic the Ab epitopes (i.e., a mimotope).

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For recognition of B cells, any part of the Αβ peptide can be used, which was shown by the example of binding of antibodies to Av with partially matching peptides. For example, peptides containing subunits 1–3, 2–6, 1–7, 4–10 and 1–11 can be used (Wagy c1 a1., 2003; Meiba c1 a1., 2002). In studies, the effect of this principle was shown in the analysis of the forms Αβ _1-11 or Αβ _1-15 . Some mismatch of these sequences, not associated with conservative substitutions, manifests itself in the functional activity and production of antibodies to Αβ. Such a discrepancy may be at least 50%, preferably at least 20%, more preferably at least 10% and more preferably 5%. The discrepancy can be between conservative variants and variants with a conservative function. Conservative polynucleotide sequence variants are variants when replacing one or more nucleotides at specific positions of the codon does not lead to a change in the encoded amino acid at that position. Variants with a conservative function are polynucleotide sequences that encode proteins in which at least one amino acid residue has been changed without changing the final conformation and function of the polypeptide, including, but not limited to, replacing an amino acid with another amino acid with the same properties (such as, for example, polarity, potential of hydrogen bonds, acidity, basicity, hydrophobicity, the presence of aromatic groups and the like). Amino acids with similar characteristics are well known in the art.

Mimeotopes are polypeptides with different sequences, but with the same three-dimensional structure, corresponding to the Αβ ₄₂ peptide, whose immunological functions are identical. Mimeotopes are any biological molecule that is unrelated to the Αβ ₄₂ structure but has identical three-dimensional antigenic epitopes and is recognized by anti-Αβ B cells. An antibody for a given antigen epitope will also recognize a mimotope that mimics this epitope. Vaccines are currently being developed that use mimotopes. Examples of some mimotopes that can be used in this way include, but are not limited to, the following.

The term alien T-cell epitopes / improper T-cell epitopes is understood to mean any foreign peptide that can cause changes in the activity of the immune system, for example, but not limited to, produce cytokines, chemokines and help B cells produce detectable titers of antibodies to Αβ ( recognizing any form of this molecule) in the serum of vaccinated people. Examples of foreign T-cell epitopes that can be used are, but are not limited to, ΡΑΌΡΕ, P2, P30, P32, P21, P23 from tetanus toxin, HbAb epitopes, HBU nuclear capsid, and influenza virus M protein. The DNA sequences of these foreign Tcellular epitopes are well known. Some difference in these sequences can lead to differences in properties. Such a difference may be at least 50%, more preferably at least 20%, and more preferably at least 10%. The discrepancy can be between conservative variants and variants with a conservative function.

Conservative polynucleotide sequence variants are variants when replacing one or more nucleotides at specific positions of the codon does not lead to a change in the encoded amino acid at that position. Variants with a conserved function are polynucleotide sequences that encode proteins in which at least one amino acid residue has been changed, while the final conformation and function of the polypeptide has not changed, including, but not limited to, the amino acid has been replaced by another amino acid with similar properties (such as, for example, polarity, potential of hydrogen bonds, acidity, basicity, hydrophobicity, the presence of aromatic groups and the like). Α amino acids with similar characteristics are well known in the art.

The term general T-cell epitopes refers to any peptide that can be recognized by a large number of ΌΚ-molecules of the immune system and cause changes in the cells of the immune system, for example, stimulate the production of cytokines, chemokines and help B-cells produce detectable titers of antibodies to Αβ in the serum of vaccinated people.

The term artificial T-cell epitopes may also be used. It refers to common T-cell epitopes, including, but not limited to, ΡΑΌΚΕ.

Molecular adjuvants are biological molecules that can modulate the immune system so that it responds to immunization with any vaccine or antigen. Molecular adjuvants can bind to receptors on the surface of immune cells or to the target portion of other molecules that are ligands that bind to the receptor. It is especially important that the receptor on the surface of immune cells is an immunomodulatory receptor. Suitable receptors on the surface of immune cells include, for example, CCK5 (for MOS), CCK6 (for β-3), GO-4P. ΙΡΝγΚ, CE21 (for C3ph and To11-like receptors (TB).

Under the term heterologous rpche / boo! Vaccination is understood as a vaccination regimen using combinations of epitope DNA vaccines or epitope vaccines based on a polypeptide / peptide / recombinant protein (i.e., a polypeptide containing an epitope recognized by B cells,

7,026,787 expressing BSC specific for Αβ, a foreign T-cell epitope (s) and molecular adjuvants), or viruses expressing only epitopes recognized by B cells that express BSC specific for Αβ, or in combination with a foreign T- cellular epitope (s) and molecular adjuvants; or viral particles containing only epitopes recognized by Cells that express VSC specific for Αβ, or in combination with foreign Tcellular epitope (s) and molecular adjuvants; or any other combination of heterologous vaccines.

In order to facilitate the introduction into the body of animal vaccines based on DNA and proteins, various methods can be used. These include, in particular, but are not limited to, the use of OS, nanoparticles, suitable adjuvants, plasters, and the like.

If DNA is used as a vaccine, then it can be delivered directly to the cell using technologies such as intramuscular or intradermal injection and subsequent electroporation, delivery using gold particles (gene gun), delivery using liposomes, or direct injection as well as other methods well known in the art. The most suitable methods for epitope DNA vaccination are, but are not limited to, the use of a gene gun and a method based on electroporation. It is with these methods that the significantly increased efficiency of introducing DNA into cells is associated, and it has been shown that their use enhances the effect of DNA vaccines by ~ 100 or more times, compared with conventional injection methods.

Positive changes in neuropathology include, but are not limited to, reduction / inhibition of soluble and insoluble Αβ deposits in the parenchyma and vascular system of the brain, decrease in stimulation with respect to glia activation, decrease in 1Ai changes in the central nervous system (in the brain and cerebrospinal fluid), inhibition / prevention death and degeneration of neurons, inhibition of cognitive impairment and / or recovery.

The recovery of cognitive functions can be measured using, for example, the cognitive function assessment scale (ΆΌΆδ-sod) and the disability assessment scale (ΌΛΌ), the Мш1-Мей1а1 5> 1ае Ехатшабой methodology - ММ8Е (Short Mental State Assessment Scale), a set of neuropsychological tests (ΝΤΒ )

By the term modifications is understood conservative variants and variants of a conservative function. Conservative variants of the polynucleotide sequence are the replacement of one or more nucleotides at predetermined codon positions, not leading to a change in the corresponding amino acid. Variants with a conservative function are polynucleotide sequences that encode proteins in which at least one amino acid residue has been changed without changing the final conformation and function of the polypeptide, including, but not limited to, replacing an amino acid with another amino acid with the same properties (such as, for example, polarity, potential of hydrogen bonds, acidity, basicity, hydrophobicity, the presence of aromatic groups and the like). Amino acids with similar characteristics are well known in the art.

By the term “vaccine composition” in the present invention is meant an immunogenic amount of an epitope vaccine against Alzheimer's disease together with a pharmaceutically acceptable carrier.

An immunogenic amount is an amount of an epitope vaccine against Alzheimer's disease sufficient to induce an immune response in the individual to whom the vaccine has been administered. The amount of vaccine administered is the amount that induces the desired immune response, the desired degree of protection against Alzheimer's disease, inhibition and stabilization of the pathology associated with Alzheimer's disease, or has a therapeutic effect. Examples of pharmaceutically acceptable carriers are, in particular, sterile pyrogen-free water and sterile pyrogen-free saline.

The vaccine composition of the present invention is suitable for patients with an early diagnosis of Alzheimer's disease. The vaccine composition developed in the framework of the present invention is also suitable for the treatment of patients with a genetic predisposition to Alzheimer's disease, for example, with a PAO mutation (a common mutation of Alzheimer's disease), a positive reaction to ApoE4, or Down's disease. In addition, the composition of the vaccine of the present invention is suitable for use in large populations that do not have Alzheimer's disease or predisposition to it, but want to protect themselves from the development of this disease.

The administration of the vaccine can be carried out by any suitable method, including parenteral injection (e.g., intraperitoneal, subcutaneous or intramuscular injection), intradermal injection, intravenous injection, nasal or airway administration. Topical administration of the vaccine via the respiratory tract can be carried out intranasally (for example, using drops, tampons or an inhaler, with which the pharmaceutical substance will be administered intranasally). Topical administration of the vaccine through the respiratory tract can also be achieved by inhalation, for example, using specially formulated respirable particles of a pharmaceutical substance (including both solid and liquid particles) containing

- 8,026,787 replicons in the form of an aerosol suspension, which will be ingested during inhalation. Methods and devices that facilitate the administration of respirable particles of pharmaceutical preparations into the body are well known, which makes it possible to choose the most suitable one from them. An immunogenic amount is the number of replicon particles that will be sufficient to induce an immune response in the object to which the vaccine has been administered. Any vaccine can be used in various combinations that are most effective for eliciting an immune response in a patient. Typically, the vaccine is administered in an amount that allows you to the right degree to induce the desired immune response. The exact amount of vaccine administered may depend on the data obtained during clinical trials.

The vaccine can be administered once, or, more preferably, repeatedly, in quantities that allow you to induce the desired immune response to the right degree. The exact schedule for administering the vaccine may depend on data obtained during clinical trials.

If the amino acid sequence of the polypeptide is at least 95% identical to the amino acid sequence of the reference peptide, it is assumed that the amino acid sequence of the polypeptide is identical to the reference sequence, despite the fact that the polypeptide sequence may contain up to five amino acid changes for every 100 amino acids of the reference protein sequence. In other words, to obtain a polypeptide whose amino acid sequence has at least 95% homology with a reference sequence, up to 5% of amino acid residues can be removed or replaced in its sequence, or up to 5% of amino acids can be inserted into the reference sequence. These changes in the reference sequence can be at the amino or carboxyl terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, and can be either individual amino acid residues randomly scattered across the reference sequence, or one or more extended groups of amino acids in reference sequence. In practice, virtually any single polypeptide that is at least 95, 96, 97, 98 or 99% identical, for example, the amino acid sequence 8EO GO N0: 2, can be precisely determined using known computer programs such as the ΒοκίΠΐ ( νίδεοηκίη Beschischpez Aia1u818 Raskade, Uegaop 8 Gog Ishkh, Oeiyesz SoshrSheg Ogoir, Ituegvuu RzszagheN Ragk, 575, §c1eise Epheus. Μαάίδοη. νίδ.53711). When using the Be8Lb program or a similar program for alignment of sequences in order to determine whether the sequence being analyzed is homologous, for example, to 95% of the reference sequence, of course, certain parameters were established in the present invention, such as the percentage of identity, which was calculated on all during the reference amino acid sequence, while up to 5% of the total number of amino acid residues in the sequence homology was allowed.

It should also be noted that the amino acid sequences mentioned in this paper are at least 90% identical to each individual site of the B-cell epitope, the foreign T-cell epitope and the molecular adjuvant sequence, and, as revealed by the analysis of a sequence selected from any of the mentioned sequences , nucleotide sequences are generally less than 90% identical, since 90% identity refers to a single fragment of the whole molecule.

The present invention presents several approaches to overcome the limitations of previous works. One of the possible approaches to overcoming the limitations noted in previous works is the regular vaccination of healthy people, or patients in the very early stages of Alzheimer's disease, which can only be diagnosed by measuring the ratios of Ριιι / Λβ and p1ai / A β in the cerebrospinal fluid and / or by screening for Αβ accumulation in the brain using a P1B-PET scan and a combination of these methods, and subsequent annual re-vaccinations. Therefore, early vaccination is very important.

Another possible approach to combat the aging of the immune system is to use preexisting memory T cells produced during life as a result of vaccination, including vaccination at school age, or as a result of infection by various pathogenic organisms. Most people already have a large number of specific memory T cells directed against specific vaccines and various pathogens. Vaccination of such people using a recombinant vaccine containing B-cell epitopes (including the humoral response to Αβ in the case of vaccination against Alzheimer's disease) and the foreign T-cell epitopes from the vaccines or pathogens mentioned below can enhance preexisting T-cell memory, stimulate their rapid differentiation into T-cell effectors, which will lead to a fast and strong immune response (antibody production) specific for B-cell epitopes of any form of β (Fig. 12). Tetanus toxin, influenza virus, and NLV are examples of such vaccines. As a result of compulsory tetanus vaccination, annual vaccination and / or infection with influenza virus and vaccination against HLV, almost every person should have CGO4 'T-cell memory specific for T-cell helper epitopes of tetanus toxin and HLV and conservative influenza virus epitopes. Therefore, re-immunization with an epitope vaccine against Alzheimer's disease containing the indicated T-cell epitopes and a strong molecular adjuvant should be sufficient to rapidly activate the preexisting T-cell memory directed against tetanus toxin, HBV and influenza virus, which leads to activation of b-cells expressing immunoglobulin receptors (BSC) to Αβ, and to strong and steady production of antibodies to Αβ in old age.

This study presents epitope vaccines against Alzheimer's disease, consisting of several copies of native B-cell epitopes, foreign T-cell epitope ΡΑΌΚΕ or multiple T-cell epitopes derived from tetanus toxin, HBV and influenza virus (such as P2, P30, P32 , P21, P23 tetanus toxin, HbAc, HBI nuclear capsid, influenza virus M protein) and molecular adjuvant (such as MES, β-β3, 3C3y). The epitope vaccine against Alzheimer's disease has improved characteristics compared to the usual vaccine against Alzheimer's, as: (ί) several copies of B-cell epitopes enhance the immune response and avidity of antibodies to antigens; (ίί) several copies of B-cell epitopes enhance the accumulation of antigens and the presentation of the corresponding peptides via APC T cells; (ίίί) an improved combination of intrinsic B-cell epitopes with epitopes of various foreign T cells can inhibit the development of tolerance and induce a stronger immune response; the replacement of A () 42 peptide epitope of native T cells with an epitope of foreign cells prevents the development of an autoreactive T-cell response; (ίίί) replacing the epitope of native T cells with the epitopes of T cells from the corresponding vaccines or pathogens that humans often exposed to, enhances the preexisting T-cell memory created over the lifetime of most people receiving the epitope vaccine against Alzheimer's disease and induces fast and strong immune response (production of antibodies to Av); (ίν) combining with a molecular adjuvant or enclosing a vaccine in it substantially enhances the immune response and shifts it in the right direction; (ν) primary immunization with an epitope DNA vaccine and subsequent enhancement of its action with an epitope vaccine based on a protein / peptide, virus-like particles or viral vectors helps to induce higher titers of antibodies to Av than with repeated immunization with a DNA vaccine.

Vaccines

This paper presents the idea of creating epitope DNA vaccines directed against Alzheimer's disease, consisting of several copies of their own B-cell epitopes, supplemented by various foreign T-cell epitopes obtained from the corresponding vaccines or as a result of infection of the patient during his life by various pathogenic organisms , and combined or mixed with a molecular adjuvant, the use of which for immunization of patients in the early stages of Alzheimer's disease, or patients with He or older who have pre-existing T cell memory, it would be safe and effective. As examples, the inventors used engineered epitope DNA vaccines and vaccines based on purified proteins for immunization of APP / TD mice and subsequent analysis of the development of pathologies and behavioral disorders.

Example 1

Immunization of APP / TD mice with an epitope DNA vaccine consisting of one or more copies of a B-cell β ₄₂ epitope, a foreign TB cell epitope, and a molecular adjuvant.

So, the inventors have developed a plasmid vaccine in which a foreign T-cell epitope (ΡΑΌΚΕ) was associated with 1 or 3 copies of the B-cell Αβ42 epitope (3Αβ _1-11 ). In addition, in this plasmid, the inventors used a macrophage cytokine (MES), which, as the inventors and other authors noted earlier, significantly enhances the T2 cell-type immune response (Vyadup c1 a1., 2001). Expression of the obtained structures ρΜΌΧΑβ _1-π- ΡΑΌΚΕ and ρΜΌΧ3Αβ _1-π -ΡΑΌΚΕ and the structure encoding an epitope vaccine without a molecular adjuvant (3Αβ _1-11 -ΡΑΌΚΕ) was observed in transfected CHO cells with the Western Blot method (Fig. 1). Epitope DNA vaccine was tested on 3-4-month-old mice of line 3. \ TD-LE. Three immunizations of line 3 mice. The TD-LE epitope DNA vaccine induced a CE4 'T cell response directed against foreign antigenic determinants, ΡΑΌΚΕ, but not against Αβ own antigen (Fig. 2). The cellular immune response was T2-oriented, as shown by flow cytometry using reagents aimed at the detection of intracellular cytokines (ΙΝΡγ and ΙΌ-4). CE4 'T-lymphocytes provide sufficient support for B cells in the production of vaccine mice with high concentrations of antibodies to 1β _1-11 . Importantly, the antibody concentration was significantly higher in mice immunized with an epitope DNA vaccine containing 3 copies of the B-cell epitope than in mice immunized with a vaccine containing only one copy (Fig. 3). In addition, polyclonal antibodies to Αβ generated by ρΜΌΧ3Αβ _1-π- ΡΑΌΚΕ had a higher avidity to Αβ ₄₂ than antibodies generated by rMES-Αβμπ-ΡΑΌΚΕ. Thus, an epitope vaccine was developed that was immunogenic in APP / TD mice.

The molecular adjuvant of MES was necessary not only for the production of higher antibody concentrations, but also for directing the humoral immune response in the direction of strict T2 specificity (the ratio 1dO1 / 1dO2s for ρ3Αβ _1-11- ΡΑΌΚΕ was 2.2, and for ρΜΌΧ3Αβ _1-11- ΡΑΌΚΕ- 10 026787

22.7). In general, analysis of the cellular and humoral immune response showed that (ί) a vaccine containing several copies of B-cell epitopes induces the production of high concentrations of β-specific antibodies with a higher avidity than a vaccine containing only one copy of epitopes; (ίί) an epitope DNA vaccine did not activate Αβ autoreactivity in T cells; (ίίί) MES molecular adjuvant additionally enhances the immune response and directs it towards anti-inflammatory immunity of the T2 type, which can be very useful for the elderly.

DNA vaccination reduced the likelihood of negative age-related changes in cognitive function in 3xTd-линии mice.

ZhTd-AE mice vaccinated with an epitope DNA vaccine showed an improvement in cognitive functions compared to naive transgenic animals of the same age. Our data showed that vaccinated mice had no difficulty in memorizing information, in contrast to unvaccinated mice, immunized animals had both short-term (1.5 h) and long-term (24 h) memory with spatial orientation. Antibodies produced after vaccination using an epitope DNA vaccine are very effective.

Further, in order to analyze the mechanism of the effect of DNA vaccination on the restoration of behavioral function disorders, the inventors analyzed neuropathological changes in immunized and control mice aged 18 ± 0.5 months. First of all, the inventors tested Αβ deposits and showed that vaccination significantly reduces the accumulation of amyloids (diffuse and hollow plaques) in various brain regions of immunized mice, compared with the control group. The epitope vaccine induced a statistically significant reduction in both soluble and insoluble forms of β ₄₂ (P <0.001) in the brain of immunized mice. The level of insoluble Αβ ₄₀ was also reduced in vaccinated animals, but its decrease was not statistically significant. Vaccination using a DNA epitope vaccine significantly reduces the pathological changes associated with inflammation (microglial activation, astrocytosis), without increasing the incidence of cerebral microhemorrhages in aging ZxTd-AE mice. The pathology of Tai in vaccinated mice is also reduced compared to control animals. In mice immunized with an epitope vaccine, there were no cases of T-cell infiltration in the brain (Moukekui s1 a1., 2008).

Example 2

Рпте-лоок! the vaccination regimen was studied on mice of the ZhTd-AE line, when the mice were immunized twice with an epitope DNA vaccine, and then its effect was enhanced by a homologous recombinant protein. The concentration of antibodies was significantly higher in mice immunized with an epitope DNA vaccine whose effect was enhanced by protein than in mice that received only an epitope DNA vaccine (Fig. 4).

Example Z.

The combination of an epitope vaccine with a molecular adjuvant enhances the immune response.

Epitope DNA vaccine coupled with molecular adjuvants of MES. β-ERZ or ZSZb induced in mice a significantly higher immune response (antibody production) than a vaccine without a molecular adjuvant (Fig. 5). Four groups of C57BB6 mice were immunized three times, with a two-week difference, with the vaccines ρM ^ C-3Aβ ₁₁ -AA ^ ΚΕ, ρβ ^ Ρ3-3Aβ ₁₁ -AA ^ ΚΕ, ρ. ^ ΑΑβιι-ΡΑΌΡΕЗЗЗб and р3Αβ ₁₁ -АА ^ ΚΕ respectively, using the gene gun. On day 10 after each immunization, serum was collected and the concentration of anti-β antibodies was measured using the EPPA method.

Example 4

Peptide epitope vaccine.

Two types of peptide-based epitope vaccines have been developed, 2Αβι _- ιι-ΡΑΌΚΕ and ΡΑΌΚΕ-2Αβι _- ιι, combined with a lysine base (2Αβι _- ιι-ΡΑΌΚΕ-ΜΑΡ or ΡΑΟΚΕ-2Αβι _- ιι-ΜΑΡ). Both peptides coupled to an OPL adjuvant induced equally high titers of anti-β antibodies in C57BB6 mice after three repeated immunizations carried out with a two-week difference (Fig. 6). To further investigate the therapeutic possibilities of the peptide epitope vaccine, 9-month-old mice of the ΑΡΡ / Т§2576 line, having moderately expressed pathological signs similar to the signs of Olzheimer's disease, were immunized with 2Αβ _1-11- ΡΑΌΚΕ-ΜΑΡ, coupled with the ΡιπΙΑ adjuvant, with the aim of the study of neuropathological changes in their brain (Chenga 5b ^ na e1 a1., 2007). Vaccination of mice line ΑΡΡ / TD2576. possessing pathological signs similar to those of Olzheimer's disease, an epitope vaccine, it helped to reduce the accumulation of the insoluble form of β, without causing any adverse effects, for example, such as the infiltration of T cells or macrophages in the central nervous system or microhemorrhages. However, such vaccination did not change the amount of the most toxic soluble oligomeric forms of Αβ. These studies suggest that preventive vaccination and / or early therapeutic vaccination can protect the body from the manifestations of Olzheimer's disease, or reduce the manifestations of this disease, while therapeutic vaccination alone cannot lead to the destruction of toxic oligomers and

- 11,026,787 can only minimally reduce existing symptoms of Alzheimer's disease.

Viral vector based vaccine.

As an alternative delivery platform for immunogenic Αβ42 B-cell epitopes, the inventors have recently developed and tested a vector based on a recombinant influenza virus that is capable of not only delivering Αβ _1- ι ₀ or any other desired peptide to the host immune system, but also providing the necessary support T cells. Recombinant influenza virus Α / Ψ8Ν / 33 (Η1Ν1) expressing B-cell epitopes опβ ₄₂ , Αβι_ι ₀ (Πη-Αβι _- | ₀ ). embedded in the HA gene, was created using the method of rescue plasmids, one of the methods of reverse genetics (8c1iskP s1 a1., 2001). C57BB / 6 mice were immunized and revaccinated twice using various combinations of attenuated live or killed viruses (Table 1).

Table 1

Monthly immunization of C57BB / 6 mice with various combinations of attenuated live or killed recombinant influenza viruses

No. Group Immunization Rimmunization one Re-immunization 2 one Pi-Αβι-ιο Live vaccine 10 ² R.H.I. ί. η. * Live vaccine 10 ⁵ p. £ .and. ί.η. The live vaccine U ^- p. £. and. ί.η. 2 ίΙυ-Αβχ-th Live vaccine as ΙΟ ² ρ.ί.ίΐ. ί .η. Live vaccine 10 ⁵ r.G.I. etc. Killed vaccine 20 mcg ί.ρ. 3 £ 1i-A | H- ₁₀ Killed vaccine 20 mcg ί.ρ. ** Killed vaccine 20 mcg ί.ρ. Killed vaccine 20 mcg ί.ρ.

* ΐ.η. - internally;

** ί.ρ. - intraperitoneally.

A recombinant influenza virus expressing Αβ _1-10 induced a rather strong immune response in all combinations, however, a stronger response was observed when the mice were immunized twice with a live attenuated virus and then the effect was enhanced by the administration of a killed vaccine (Fig. 7). All groups of mice immunized with the recombinant virus predominantly induced antibodies of the IgO2b and IgO2b isotypes. The ratio 1dO1 / 1dO2a ^b was less than 1, which means that the inventors induced a predominantly Ty cell-type immune response. The response of the YOU-cell type should not induce undesirable side effects, since it is specific for the influenza virus and not for собственнымβ own antigens.

Protein epitope vaccine. Protein epitope vaccines ΜΌί'-Αβπ-ΡΑΌΚΕ and ΜΌί'ΆΑβπΡΑΌΚΕ, which did not contain any auxiliary substances, induced a strong immune response after immunization with C57BB6 mice using them (Fig. 8), however, the immune response induced by a vaccine carrying three copies of B -cellular epitopes was significantly stronger than that induced by a vaccine containing only one copy of B-cell epitopes. For the production of эп ^ 3Αβ _1-π- ΡΑΌΡΕ and ΜΌ ^ Αβμπ-бел protein epitope vaccines, minigens were subcloned into the ρΕΤ24ά expression vector for E.coB at the C-terminal position of the Ηίδ-Tad protein in order to generate recombinant proteins in E.coB. Recombinant proteins were isolated from E. coB strains transformed with ρΕΤ-ΕΤ ^ 3Αβ _1-π -ΡΑΌΡΕ or ρΕΤ-Μ ^ С-Сβ _{| - ||} -ΡΑ ^ ΡΕ. using columns ΝίΝΤΑ, and then purified using a gel that removes endotoxins. Protein purity was analyzed by electrophoresis in a 10% Ττίδ-δΌδ-еле gel. Protein bands were visualized by gel staining with Coomassie Blue Dye. Mice received 50 mg of protein subcutaneously, four times, with a frequency of two weeks, serum was collected on the 10th day after each immunization.

Example 5

Testing for possible preexisting T-cell memory.

1. Mice were immunized with a DNA vaccine encoding an epitope of ΡΑΌΡΕ T cells of helper cells and containing a molecular adjuvant of MES. After a 3-month rest period during which ΡΑΌΡΕ-specific T-cell memory was to be formed, immunization of mice was enhanced by an epitope DNA vaccine encoding мини ^ 3Αβ _1-11 -ΡΑΌΡΕ minigens. The control group of mice, without preexisting T-cell memory, was immunized only with an epitope DNA vaccine, after which the immune response was analyzed in both groups (production of antibodies to Αβ). Mice with pre-existing T-cell memory produced significantly higher concentrations of anti-β antibodies than mice without T-cell memory (Fig. 9).

2. The inventors constructed and tested on mice of the C57Bbb line an epitope DNA vaccine encoding three copies of the Av ₄₂ (3Av _p ) B-cell epitope and eight epitopes of Helper cells obtained from the corresponding vaccines, and including the molecular adjuvant MES (ρΜΌί '' -3Αβ || -Τ1ι) (Fig. 11). Two groups of mice were immunized with the rMES-Zavc-T vaccine or control vector. On the 7th day after the last immunization, the mice were euthanized, but before that they had taken blood to study the cellular and humoral immune response. In particular, the inventors investigated the proliferation of ί'Ό4 'T cells and the production of cytokines 1b-4, ΙΡΝγ (Fig. 10) using the PAC8 method. Stimulation of splenocytes of mice immunized with rMES-ZAvp-TB using a mixture of all eight proteins induced pronounced proliferation of T cells. It is important to note that CE4 + T cells of immunized mice were activated strongly by P32 or P21 peptides, medium by P23 and HBU19-33 peptides, and weakly by ΙΝΡ17-31. rMES-3Av ₁₁ -TB induced pronounced proliferation of T cells specific for different epitopes of Ty cells obtained from the corresponding vaccines and did not activate the immune response of T cells to β. Splenocytes from mice that received the control vector did not proliferate after repeated stimulation with Tykletok or Αβ ₄₀ epitopes. The analysis of ΙΡΝγ (Ty1-cell type cytokine) and 1b-4 (Ty2-cell cytokine) in immune and control splenocytes fully complies with these data: (ί) the ρΜΌί'ΆΑβπTy vector activates both ΙΡΝγ and 1b-4 production in CE4 + T -cells after their repeated stimulation with ίη νίίτο peptides P32, P21 and ΙΝΡ17-31, but not with Αβ ₄₀ (Fig. 10). The vector ρΜΌί'-3Αβ 11-Th induces a strong humoral immune response in mice (138.9 ± b8.9 μg / ml).

Example b.

Amyloid fibrils can be recognized by the mature immune system as foreign, since during the development of the immune system they are absent in the body. Using rabbit immune serum directed against oligomeric forms of Αβ ₄₂ , the inventors screened a peptide library of phages to identify foreign conformational mimotopes of Αβ. The immune serum of both animals primarily recognized proteins with an EPKN motif in the Αβ ₄₂ sequence, but it also recognized several phage clones containing mimic epitopes (mimotopes)

Claims (17)

1. A nucleic acid construct encoding a polypeptide that induces an immune response to a human b-amyloid peptide (AB) and contains a B-cell epitope that includes one or three copies of a fragment of AB _1-11 , a T-cell epitope that is foreign to relative to humans, the T helper epitope ΡΑΌΚΕ, and a molecular adjuvant, the nucleotide sequence of the construct is at least 90% homologous to the sequence selected from the group consisting of 8ΕΟ GO N0: 11, 12, 14 and 15, and the amino acid sequence of the decree of the polypeptide is at least 90% homologous to the sequence selected from the group consisting of δΕΟ GO N0: 26, 27, 29 and 30. 2. The construction according to claim 1, where the immune response is a humoral or cellular immune response. 3. The construct according to claim 1, where the specified foreign T-cell epitope is connected to the C-end of the extracellular epitope. 4. The construction according to claim 1, where the specified molecular adjuvant is connected to the Ν-terminus or to the end of the encoded polypeptide. 5. The construction according to claim 1, the sequence of which is selected from the group consisting of 8Ε0 GO N0: 11, 12, 14 and 15. 6. The construction according to claim 1, where the molecular adjuvant is selected from the group consisting of MES, v-ER3, 3C3L. 7. An expression vector containing the nucleic acid construct according to claim 1, capable of inducing a humoral response to Av. 8. The plasmid containing the nucleic acid construct according to claim 1. - 40,026,787 9. The method of inducing an immune response in animals and humans by introducing an effective amount of a nucleic acid construct according to claim 1. 10. The method according to claim 9, where the specified animal and man receive the specified design to establish a diagnosis of Alzheimer's disease. 11. The method according to p. 10, where the specified animal and man receive the specified design in the early stages of Alzheimer's disease. 12. The method according to claim 10, where the specified animal and man receive the specified design after establishing a diagnosis of Alzheimer's disease. 13. A method of inducing a cellular and humoral immune response in animals and humans, comprising conducting primary immunization with the construct of claim 1 and then enhancing its effect by introducing a polypeptide encoded by the construct of claim 1. 14. The method according to item 13, where the humoral immune response is the production of antibodies specific for any form of Av (monomeric, oligomeric, fibrillar). 15. The method according to item 13, where the cellular immune response is specific for the T-helper epitope of RAOKE. 16. A method of treating Alzheimer's disease, comprising introducing into the patient’s body an effective amount of the nucleic acid construct according to claim 1 using a needle, a gene gun, electroporation, a pneumatic syringe Vu] ec1og, plasters and other delivery devices. 17. A method of treating Alzheimer's disease, comprising introducing into the patient's body an effective amount of the nucleic acid construct according to claim 1 simultaneously and / or sequentially with any suitable adjuvant using any delivery method.