UA85815C2 - Use of immunogene for prevention and treatment of alzheimer's disease or other diseases associated with deposition of amyloid - Google Patents

Abstract

. Disclosed are novel methods for combatting diseases characterized by deposition of amyloid. The methods generally rely on immunization against amyloid precursor protien (APP) or beta amyloid (Abeta). Immunization is preferably effected by administration of analogues of autologous APP or Abeta, said analogues being capable of inducing antibody production against the autologous amyloidogenic polypeptides. Especially preferred as an immunogen is autologous Abeta which has been modified by introduction of one single or a few foreign, immunodominant and promiscuous T-cell epitopes. Also disclosed are nucleic acid vaccination against APP or Abeta and vaccination using live vaccines as well as methods and means useful for the vaccination. Such methods and means include methods for the preparation of analogues and pharmaceutical formulations, as well as nucleic acid fragments, vectors, transformed cells, polypeptides and pharmaceutical formulations.

Description

Influence of AO

AR is the most common cause of dementia in people aged 65 and older. It is a major health problem due to its enormous impact on individuals, families, the health care system, and society in general. Scientists estimate that more than 4 million people currently suffer from this disease, and the frequency of the disease in those individuals who are over 65 years old doubles every 5 years. It is also estimated that approximately 360,000 new cases (incident) will occur each year, although this number will increase as the population ages (VgooKteueg ei a!., 1998).

AI imposes a heavy economic burden on society. A recent study in the United States estimated that the annual cost of treating one patient with AO was 518,408 for a patient with mild

AYU, 530096 - for a patient with moderate AU and 536132 - for a patient with severe AU. It is estimated that the annual US government expenditure on treating patients with AO is just over $550 billion (Geop et al., 1998).

Approximately 4 million Americans are 85 years of age or older, and in the most industrialized countries this age group represents one of the fastest growing segments of the population. It is estimated that this group will number approximately 8.5 million in the US by 2030; some experts who study population changes suggest that this number may even be higher. As more and more people live longer, the number of people affected by age-related diseases, including AO, will continue to increase. For example, some studies show that about half of all people aged 85 and older have some form of dementia (Maiyopai! Ip5icie op Adipd

Rgodgezv VNeroigi op AI2Peiitegz Oivzease, 1999).

Main characteristics of AO

Two abnormal structures in the brain are hallmarks of AO: amyloid plaques and neurofibrillary tangles (METs). Plaques are dense, almost completely insoluble accumulations of protein and cellular matter outside and around brain neurons. Tangles are insoluble interwoven fibers that accumulate inside neurons.

There are two forms of AO: familial AJ (BA), which is subject to a certain type of inheritance, and sporadic AS, for which no obvious type of inheritance is observed. Because of differences in disease onset, AO is further described as early-onset (occurring in a person younger than 65 years) and late-onset (occurring in a person 65 years or older). Early-onset AO is rare (about 10 percent of cases) and usually affects people between the ages of 30 and 60.

Some early-onset forms of A are hereditary and affect family members. Early-onset AO also often progresses more rapidly than the more common late-onset form.

All GAOs known so far are characterized by an early onset, and nowadays it is known that up to 50 percent of GAO cases are caused by defects in three genes located on three different chromosomes.

They are mutations in the APP gene on chromosome 21; mutations in a gene called presenilin-1 on chromosome 14 and mutations in a gene called presenilin-2 on chromosome 1. However, there is still no evidence that any of these mutations play a major role in the more frequent, sporadic or non-familial form of AO with late onset (Maiiopai! Ipziyshe op Adipd Rgodge55 Kerogpi op AI2peitegz Oizease, 19991.

Amyloid plaques

In AO, amyloid plaques first develop in areas of the brain associated with memory and other cognitive functions. They consist of almost completely insoluble accumulations of beta-amyloid (hereinafter referred to as AD), which is protein fragments of a larger protein called the amyloid precursor protein (APP, the amino acid sequence of which is given below in ZEO

IO MO:2), mixed with parts of neurons and non-neuronal cells such as microglia and astrocytes.

It is not known whether amyloid plaques themselves are the primary cause of AO or are a byproduct of the AO process. Certainly, as shown for the inherited form of AO caused by mutations in the APP gene, changes in the APP protein can cause AO, and the formation of Aβ plaques is closely associated with the progression of the disease in humans. 19981.

APP

APP is one of many proteins associated with cell membranes. After its formation, APP is embedded in the nerve cell membrane, being located partly inside and partly outside the cell.

Recent studies using transgenic mice have shown that apparently APP plays an important role in the growth and survival of neurons. For example, certain forms and amounts of APP can protect neurons from both short-term and long-term damage, and can put damaged neurons in a state with a better ability to self-repair and help parts of neurons grow after brain injury.

While APP is embedded in the cell membrane, proteases act on special areas of APP, cleaving it into protein fragments. One protease helps cleave APP to form AD, while another protease cleaves APP in the middle of the amyloid fragment so that AD cannot form. The Ar fragment is formed from two different parts, a shorter AD with a length of 40 (or 41) amino acids, which is relatively soluble and slowly aggregates, and a slightly longer "sticky" AD, with a length of 42 amino acids, which quickly forms insoluble aggregates. While AD is formed, it is not yet known exactly how it moves through or around nerve cells. At the final stage of the process, "sticky" AD aggregates into long filaments from the outside of the cell and, together with fragments of dead or dying neurons and microglia with astrocytes, forms plaques that characterize AD in brain tissue.

There is some evidence that mutations in APP become more likely when AD is cleaved from the APP precursor, thus causing the formation of either large amounts of total AD or relatively large amounts of the "sticky" form. They also found that mutations in presenilin genes can contribute to neuronal degeneration in at least two ways: by modifying AD production or by triggering cell death more directly. Other studies suggest that mutated presenilins 1 and 2 may be involved in the increased rate of apoptosis.

Ar is believed to be toxic to neurons. In tissue culture studies, the researchers found increased death of hippocampal neuronal cells engineered to overexpress mutant forms of human APP compared to neurons overexpressing normal human APP (oo ei ai.,

In addition, increased expression or expression of modified forms of the AD protein, as shown in animal models, causes symptoms similar to those of Alzheimer's disease.

Based on evidence that increased formation of Aβ, its aggregation into plaques, and resulting neurotoxicity can lead to Aβ0, investigation of conditions in which Aβ aggregation into plaques can be reduced or even blocked is of therapeutic interest.

Presenilins

Mutations in presenilin-1 (5-180) account for nearly 5,095 of all cases of early-onset familial AO (RAB). About 30 mutations leading to AI have been identified. The onset of AO varies, depending on the mutations. Mutations in presenilin-2 are responsible for a much smaller proportion of RAJ cases, but are nevertheless a significant factor. Whether presenilins are involved in sporadic non-familial AO is unknown. The function of presenilins is unknown, but they appear to be involved in the processing of APP to produce Ar-42 (a longer sticky form of the peptide, ZEO IU MO:2, residues 673-714), since patients with AO with mutations in presenilin have increased levels of this peptide. Whether presenilins also play a role in inducing MTE formation is unclear. Some data suggest that presenilins may also have a more direct role in the degeneration and death of neurons. Presenilin-1 is located on chromosome 14, while presenilin-2 is linked to chromosome 1. If a subject carries a mutated version of at least one of these genes, he or she is almost certain to develop early-onset AO.

It is somewhat unclear whether presenilin-1 is identical to the hypothetical γ-secretase involved in APP processing

IMagive eyi a!., 1998).

Apolipoprotein E

Apolipoprotein E is usually associated with cholesterol, but it is also found in plaques and tangles in the brain in AJ. Despite the fact that alleles 1-3 are considered not to be involved in Ab0, there is a significant correlation between the presence of the AROBE-e4 allele and the development of AO with a late onset (Zygitaktsneg ei a!yi., 1993.

However, it represents a risk factor rather than a direct cause as in the case of presenilin and APP mutations, and it is not limited to familial AJ.

The pathways by which APOE4 protein increases the likelihood of developing AO0 are not known for sure, but one possible theory is that it facilitates the accumulation of Aβ, and this contributes to a reduction in the age of onset of AO, or the presence or absence of specific alleles AROE can affect the way neurons respond to damage (Vitsipi et al., 19991.

It has been shown that Aro At is also amyloidogenic. Intact Aro A1 ip migo can independently form amyloid-like fibrils, which are stained with Congo red (At U. Rayoi. 147 (2): 238-244 (Atsa. 1995),

Umizpiemuzki T., sSoiarek A.A., Kida E., MMizpiemuzKi K.E., Rgapdiope V.|.

Apparently, there are conflicting results indicating that positive allele effects do occur

AROB-e4 in relation to the reduction of symptoms of mental loss in comparison with other alleles of I5Yet, Vgapayi, 1997, Appai5oi MeigoYodu 411.

Neurofibrillary tangles

This second sign of AI is an abnormal accumulation of twisted threads found inside nerve cells. The main component of the tangles is a form of protein called tau (1). In the central nervous system, tau bileus are best known for their ability to bind to microtubules, which are one of the components of the cell's internal support structure, or cytoskeleton, and help stabilize them. However, in AO, tau proteins are chemically altered, and such altered tau proteins are no longer able to stabilize microtubules, causing their disintegration. This destruction of the transport system can first lead to disruption of connections between nerve cells, and later can lead to the death of neurons.

In AO, chemically altered tau proteins intertwine into paired spiral filaments, which are two threads of tau proteins wound around each other. These filaments are the main substance found in neurofibrillary tangles. One recent study found neurofibrillary changes in less than 6 percent of neurons in a defined part of the hippocampus in healthy brains, more than 43 percent of those neurons in people who died of mild AOS, and 71 percent of those neurons in people who died from severe AO. When studying the death of neurons, they found a similar progression.

Evidence of this type supports the idea that the formation of tangles and the death of neurons progress together in the process of AO (Maiyopai Ipzishe op Adipd Rgodgez55 Kerogpi op AI2peiiteg5 Oizease, 19991.

Taupathies and tangles

Severe neurodegenerative diseases other than AO are characterized by the aggregation of tau proteins into insoluble filaments in neurons and glia, leading to dysfunction and death. More recently, several groups of researchers who studied families with different types of inherited mental retardation, different from AI, discovered the first mutation in the tau protein gene on chromosome 17 (Siagk et al., 1998; Niyop et al., 1998; Roogkai ei ai., 1998; 5ripapinpi ei a!., 19981). In these families, mutations in the tau protein gene cause neuronal death and dementia.

Such disorders, which share some of the characteristics with AO, but differ in some important aspects, are collectively called "frontotemporal dementia and parkinsonism linked to chromosome 17" (ETOR-17). They are diseases such as Parkinson's disease, some forms of amyotrophic lateral sclerosis (A!5), corticobasal degeneration, progressive supranuclear palsy, and Pick's disease, all of which are characterized by abnormal accumulation of tau protein.

Other neurological diseases similar to AI

There are important parallels between AO and other neurological diseases, including prion diseases (such as kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalitis), the disease

Parkinson's disease, Huntington's disease and frontotemporal dementia. All of them are associated with the accumulation of abnormal proteins in the brain. Both AO and prion diseases cause dementia and death, and are associated with the formation of insoluble amyloid fibrils, but from distinct membrane proteins.

Scientists studying Parkinson's disease, the second most common disorder after AO, have identified the first gene linked to the disease. This gene encodes a protein called synuclein, which, interestingly, is also found in amyloid plaques in the brains of patients with AO (Hamedap C, 1998, Sepote Kezv. 8 (9): 871-80). The researchers also found that genetic defects in Huntington's disease, another progressive neurodegenerative disease that causes dementia, cause the protein

Huntington's forms insoluble fibrils, which are very similar to AD fibrils in AO and protein fibrils in the primary disease I (zspeggipdeg E, ei aI, 1999, RMABZ Ts.5.A. 96 (8): 4604-9).

The scientists also discovered a new gene that, in a mutant form, is responsible for familial English dementia (EFD), a rare inherited disease that causes severe motor impairment and progressive dementia similar to that seen in A. Biochemical analysis of amyloid fibrils found in plaques in EVO, a new peptide called Abgi was found (Mida! ei ai!., 1999). A mutation at a specific point in this gene leads to the production of a longer than usual Vii protein. The Abgi peptide, which is cut off from the mutant end of the Bgi protein, is deposited in the form of amyloid fibrils. Such plaques are believed to lead to the neuronal dysfunction and dementia that characterize EVO.

Immunization of Ar

The immune system is usually involved in the clearance of foreign proteins and protein particles in the body, but the accumulations associated with the above diseases mainly consist of self-proteins, so the interpretation of the role of the immune system in the control of these diseases is less obvious. In addition, the accumulations are located in compartments (CNS) normally separated from the immune system, both of which suggest that any vaccine or immunotherapeutic approach will be unsuccessful.

However, scientists recently tried to immunize mice with a vaccine consisting of heterologous human AD and a substance known as an immune system stimulator (Zspepk ei ai!., 1999 and UMO 99/27944|.

The vaccine was tested in a specific model of AI in transgenic mice with a mutant human APP gene inserted into mouse DNA. As mice aged, modified APP protein was produced and amyloid plaques developed. This mouse model was used to test the possible effect of vaccination against modified transgenic human APP on plaque formation. In the first experiment, one group of transgenic mice was given monthly injections of the vaccine, starting at the age of b weeks and ending at the age of 11 weeks. The second group of mice did not receive injections and served as a control group. By 13 months of age, mice in the control group had plaques covering 2 to 6 percent of their brains.

In contrast, immunized mice had virtually no plaques.

In the second experiment, the scientists started injecting at 11 months, when some plaques had already developed. After 7 months, control transgenic mice had 17 times more plaques in the brain, while mice receiving the vaccine had a 9995-fold reduction in plaques compared to 18-month-old control transgenic mice. It was found that in some mice, some of the previous plaque deposits were removed as a result of the treatment. They also found that other damage associated with the plaques, such as inflammation and abnormal processes in nerve cells, was reduced as a result of immunization.

Thus, the above is a preliminary study in mice, and for example, scientists need to find out whether the vaccinated mice remain healthy in other ways and whether the memory of the vaccinated mice is preserved normally. Moreover, since the mouse model does not fully reflect

AR (animals do not develop neurofibrillary tangles, and a large number of their neurons do not die), more research is needed to determine whether a human has a mouse-like response or a response different from that of a mouse. Another issue to consider is that the method may be able to "treat" the amyloid build-up but not stop dementia from developing.

Technical problems also represent great difficulties. For example, it is unlikely, if at all possible, to use this method to create a vaccine that makes it possible to raise the level of antibodies against one's own proteins in a person. Thus, numerous safety and efficacy issues must be resolved before any human trials can be considered.

Thus, the work of bspepkK ei a. shows that if you can induce a strong immune response to the self-proteins in protein deposits in the central nervous system, such as the plaques that form in AB, you can both prevent the build-up and eliminate the plaques that have already formed.

Recently, clinical trials using the AD vaccines discussed above have been limited by adverse effects: a number of vaccinated subjects developed chronic encephalitis, which may have resulted from uncontrolled autoimmunity against AD in the CNS.

The purpose of this invention is to provide new ways of treating conditions such as AO characterized by the accumulation of amyloid. An additional goal is to develop an autovaccine against amyloid to develop a new way to treat AO and other pathological disorders associated with amyloid deposition.

The essence of the invention

The application of autovaccination method to generate strong immune responses against otherwise non-immunogenic APP and AD is described here. The preparation of such vaccines for the prevention, possible treatment or alleviation of symptoms of diseases associated with amyloid deposits is also described.

Thus, in its broadest and most comprehensive scope, the present invention relates to a method of down-regulating amyloid precursor protein (APP) or beta-amyloid (AD) ip mimo in animals, including humans, wherein the method includes effective presentation to the animal's immune system an immunogenically effective amount of at least one analog of APP or AD, which contains in one molecule at least one B-cell epitope of APP and/or Ar and at least one foreign T-helper epitope (Tn-epitope), so that immunization of an animal with this analog causes the production of antibodies against analogs of APP or AD of an animal, where analog a) is a polyamino acid consisting of at least one copy of the subsequence of residues 672-714 in ZEO IO MO:2, where a foreign Tn-epitope is introduced by means of addition and/or insertion, and/or deletion , and/or amino acid substitutions, where the subsequence is selected from the group consisting of residues 1-42,

residues 1-40, residues 1-39, residues 1-35, residues 1-34, residues 1-28, residues 1-12, residues 1-5, residues 13-28, residues 13-35, residues 17-28, residues 25-35, residues 35-40, residues 36-42 and residues 35-42 of the amino acid sequence, consisting of amino acid residues 673-714 5EO IU MO:2; and/or p) is a polyamino acid containing a foreign Tn-epitope and a broken APP sequence or

AD, so that the analogue does not contain any subsequence 5EO OO MO:2, which productively binds to MHC molecules of class II, initiating a T-cell response; and/or c) is a polyamino acid that includes a foreign Tn epitope and amino acids derived from APP or AD, and includes 1 single methionine residue located at the C-terminus of the analog, and other methionine residues in APP or Ar and in the foreign Tn-epitope are replaced or deleted and preferably replaced by leucine or isoleucine; and/or a) is a conjugate that includes a polyhydroxypolymer backbone, to which a polyamino acid as defined in a) is separately attached, and/or a polyamino acid as defined in b), and/or a polyamino acid as defined in c ); and/or e) is a conjugate comprising a polyhydroxypolymer backbone to which 1) a foreign Tn epitope and 2) a polyamino acid selected from the group consisting of subsequences as defined in a) of the interrupted sequence are separately attached APP or Ар, as defined in B), and an amino acid sequence derived from APP or AD, including 1 single methionine residue located at the C-terminus, and other methionine residues in APP or Ар and in the foreign Tn-epitope are substituted or removed and preferably replaced by leucine or isoleucine.

The assignee of this invention previously filed an international patent application relating to safe vaccination strategies against amyloidogenic peptides, such as APP or AVD, cf. (MO 01/622841).

This application was not published at the time of the filing date of this application and, in addition, does not contain details related to the above-mentioned suitable analogues of APP and AD.

The invention also relates to analogues of APP and Ar, as well as to fragments of nucleic acids encoding their subset. The invention also relates to immunogenic compositions that include analogs or fragments of nucleic acids.

Figure 1: Schematic representation of Ashomas variants extracted from the amyloid precursor protein to elicit responses with antibodies against the AD protein Ar-43 (or C-100). APP is shown schematically at the top of the figure, and the remaining schematic designs show that model epitopes of Pa2 and RZO replace or insert into various truncated APP products. In the Figure, the black block represents the signal sequence of APP, the oblique hatching in two directions means the extracellular region of APP, the dark vertical hatching corresponds to the transmembrane domain of APP, the light vertical hatching corresponds to the intracellular domain of APP, the bold oblique hatching means the RZO epitope and the thin oblique hatching means the P2 epitope. A frame of solid lines means Ar-42/43, and a frame of solid lines together with a frame of dashed lines means S-100. "Areia" means Ar.

Figure 2: Schematic representation of the synthesis of immunogenic conjugates used in most cases. Peptide A (any antigenic sequence, such as the AD sequence described herein) and peptide B (an amino acid sequence including a T-helper epitope) are synthesized and mixed. After they come into contact with the corresponding activated polyhydroxy polymer, the peptides

A and B are joined by means of an active group in a ratio corresponding to the initial ratio between these two substances in the peptide mixture. For details, see example 4.

Definition

Next, a number of terms used in this description and claims to explain the limits of the invention will be defined and explained in detail.

The terms "amyloid" and "amyloid protein" as used herein are used interchangeably and refer to a class of proteinaceous unbranched fibrils of indefinite length. Amyloid fibrils exhibit the ability to stain with Congo red and have a folded p-structure in which polypeptide chains are organized into p-layers. Amyloid typically originates from amyloidogenic proteins that have widely differing precursor structures, but all of which can undergo structural conversion into an aberrantly folded form that is the building block of the D-layer helix protofilament. Usually, the diameter of amyloid fibrils varies from approximately 70 to 120A.

The term "amyloidogenic protein" is intended to refer to a polypeptide that is involved in the formation of amyloid deposits, or is essentially part of the deposits or is part of a biosynthetic pathway leading to the formation of deposits. So, APP and AD are examples of amyloidogenic proteins, but proteins involved in their metabolism can also be considered amyloidogenic proteins. "Amyloid polypeptide" is intended herein to refer to polypeptides comprising the amino acid sequence of the amyloidogenic proteins discussed above, derived from humans or other mammals (or truncated variants thereof that share with the intact amyloidogenic protein a substantial amount

B-cell epitopes), therefore, the amyloidogenic polypeptide may, for example, include a substantial part of the precursor of the amyloidogenic polypeptide (in the case of AD, one of the possible amyloid polypeptides may originate from APP). The term also includes non-glycosylated forms of amyloidogenic polypeptides obtained in prokaryotic systems, as well as forms with different glycosylation profiles through the use of, for example, yeast or other non-mammalian eukaryotic expressing systems. However, it should be noted that when the term "amyloidogenic polypeptide" is used, it means that the polypeptide in question, when presented to exposed animals, is usually non-immunogenic. In other words, an amyloidogenic polypeptide is a self-protein or an analog of such a self-protein that normally does not initiate an immune response against the amyloidogenic protein of the animal in question. "Analog" is a molecule derived from APP or AD containing one or more changes in its molecular structure. Such a change, for example, can be made by fusion of APP or AD polyamino acids with a suitable partner for fusion (i.e., a change in the primary structure, which exclusively includes C - and/or

M-terminal addition of amino acid residues) and/or can be carried out in the form of insertions and/or deletions and/or substitutions of the amino acid sequence of the polypeptide. The term also includes derivatized molecules obtained from APP or Ар, cf. discussion of APP or AD modifications below. In some cases, the analog can be engineered to reduce or even eliminate the ability to elicit antibodies against the normal amyloid precursor protein(s), thereby avoiding unwanted interaction with the (physiologically normal) non-aggregated form of the amyloid precursor polypeptide .

It should be noted that the use of a foreign analog (eg, a canine or porcine analog) of human APP or AD as a human vaccine is expected to produce the desired immunity against APP or

HELL. Such application of a foreign analogue is also considered as part of the invention.

The term "polypeptide" in this context is intended to denote both short peptides of 2 to 10 amino acid residues, and oligopeptides of 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. Moreover, it is also assumed that this term covers proteins, that is, functional biological molecules that include at least one polypeptide; when such biological molecules contain at least two polypeptides, the latter may form complexes, be covalently linked or may be non-covalently linked. The polypeptide(s) in the protein may be glycosylated and/or lipoylated, and/or include prosthetic groups. The term "polyamino acid" is also equivalent to the term "polypeptide".

The terms "T-lymphocyte" and "T-cell" are used interchangeably for thymus-derived lymphocytes that are responsible for various types of cell-mediated immune responses as well as helper activity in the humoral immune response. Similarly, the terms "B-lymphocyte" and "B-cell" are used interchangeably for lymphocytes that produce antibodies.

The term "subsequence" means any continuous stretch of at least 3 amino acids or, when appropriate, at least 3 nucleotides, directly derived from the natural amino acid sequence or amyloid nucleic acid sequence, respectively.

The term "animal" in this context, as a rule, is intended to denote a species of animal (mostly a mammal), such as Noto 5ariep5, Sapi5 dotezviisis, etc., and not just a single animal. However, this term also refers to a population of such an animal species, since it is important that all individuals immunized by a method according to the present invention possess essentially the same APP or AD, when taking into account the immunization of the animals with the same immunogenic agent(s). . Experts understand that an animal in this context is a living being with an immune system. Preferably, the animal is a vertebrate, such as a mammal.

By the term "downregulation of APP or AD ip mimo" here is meant a reduction in the living organism of the total amount of accumulated amyloid protein (or amyloid as such) of the corresponding type.

Down-regulation can be achieved by several mechanisms, of which simple interaction with amyloid by binding antibodies, so as to prevent abnormal aggregation, is the simplest. However, it is also within the scope of this invention that the binding of antibodies leads to the removal of amyloid by engulfing cells (such as macrophages and other phagocytic cells) and that the antibodies interact with other amyloidogenic polypeptides that lead to the formation of amyloid.

An additional possibility is that antibodies bind AD outside the CNS, thus removing AD from the CNS using a simple principle of mass action.

The expression "effective presentation ... to the immune system" is intended to indicate that the immune system of animals in a controlled manner undergoes an immunogenic provocation of an immune response. As will become clear from the description below, such a provocation of the immune system response can be carried out in a number of ways, the most important of which are vaccination with a polypeptide containing "pharmacines" (ie, a vaccine administered to treat or improve the condition of an ongoing disease) or vaccination nucleic acid containing "pharmacins". An important result to achieve is that the immunocompetent cells of the animal oppose the antigen in an immunologically effective manner, while the exact way of achieving such a result is less important to the idea according to the invention underlying the present invention.

The term "immunogenically effective amount" has a meaning generally accepted in the art, that is, it means an amount of immunogen that is sufficient to induce an immune response, which significantly includes pathogens that share common immunological properties with the immunogen.

When it is said that APP or AD is "modified", here it means that chemical modification of the APP or Aβ polypeptide was carried out. Such a modification may be the derivation (for example, alkylation) of certain amino acid residues in the sequence, but as will be understood from the description below, preferred modifications include changes in the primary structure of the amino acid sequence.

When "autotolerance to APP or AB" is discussed, it is understood that since the polypeptide is the own protein of the population to be vaccinated, no immune response against the polypeptide occurs in normal individuals in the population; however, this cannot be ruled out because rare individuals in the animal population may be able to produce antibodies to the native polypeptide, for example, as part of an autoimmune disorder. At least, an animal is usually autotolerant only to its own APP or AD, but it cannot be excluded that a given animal will also be tolerant to analogues obtained from another animal species or from a population with a different phenotype. "Foreign T-cell epitope" (or: "Foreign T-lymphocyte epitope") is a peptide capable of binding to the MH molecule and stimulating T-cells in an animal species. The preferred foreign T-cell epitopes are "mixed" epitopes, that is, epitopes that are bound by the main part of MHC molecules of a particular class in an animal species or population. Only a very limited number of such mixed T-cell epitopes are known and are discussed in detail below. Mixed T-cell epitopes are also referred to as "universal" T-cell epitopes. It should be noted that for the immunogens used in accordance with the present invention to be effective as possible in the largest part of the animal population, it may be necessary to 1) insert several foreign T-cell epitopes into the same analogue or 2) obtain several analogues where each of the analogues has a different inserted mixed epitope. It should also be noted that

that the idea of foreign T-cell epitopes also includes the use of hidden T-cell epitopes, that is, epitopes derived from self-proteins that cause an immunological response only when they exist in an isolated form, not being part of the self-protein in question. "Foreign T-helper lymphocyte epitope" (foreign Tn-epitope) is a foreign T-cell epitope that binds to the MHC class II molecule and that can be presented on the surface of the epitope-presenting cell (ARC) in the bound to a class II type MH molecule. "Functional region" of a (biological) molecule in this context means a region of the molecule that is responsible for at least one of the biochemical or physiological effects caused by the molecule. It is well known in the art that many enzymes and other effector molecules have an active site that is responsible for the effects caused by the molecule in question. Other regions of the molecule may serve stabilization or solubility enhancement purposes and may therefore be removed if these purposes are not relevant in the context of a particular practice of the present invention. For example, in APP or AR, it is possible to use defined cytokines as modifying groups (cf. detailed discussion below), in which case the stability problem may not be significant, since binding to APP or AD may provide the necessary stability.

The term "adjuvant" has the same meaning that is generally accepted in the field of vaccine technology, that is, a substance or composition of substances that 1) is not capable of independently eliciting a specific immune response against the vaccine immunogen, but which, 2) nevertheless is capable of enhancing the immune response against immunogen. Or, in other words, vaccination with a single adjuvant does not elicit an immune response against the immunogen, vaccination with an immunogen may or may not initiate an immune response against the immunogen, but combined vaccination with an immunogen and adjuvant elicits an immune response to the immunogen that is stronger than caused by one immunogen. "Positioning or targeting" of a molecule in this context is intended to refer to a situation where the molecule, when administered to an animal, preferentially appears in a specific tissue(s) or is preferentially associated with specific cells or cell types. The effect can be achieved in a number of ways, including forming the molecule into a composition that facilitates positioning, or introducing groups into the molecule that facilitate positioning. These problems are discussed in detail below. "Stimulation of the immune system" means that the substance or composition of substances exhibits a common, non-specific immunostimulatory effect. A number of adjuvants and putative adjuvants (such as certain cytokines) have the ability to stimulate the immune system. The result of the use of immunostimulating agents is an increased "readiness" of the immune system, which means that joint or subsequent immunization with an immunogen causes a much more effective immune response compared to a separate application of the immunogen. "Productive binding" means the binding of the peptide to the MHC molecule (class I or II) so that it is possible to stimulate T cells that bind to cells that present the peptide bound to the MHC molecule. For example, a peptide bound to a class I MHC molecule! on the surface of APC, is productively bound if this APC stimulates T cells that bind to the presented peptide-MNO class I complex.

Preferred implementation of down-regulation of amyloid It is preferable that the analogue used as an immunogen in the method according to the invention is a modified molecule of APP or AD, where there is at least one change in the amino acid sequence of APP or AD, because in this case the chances of obtaining a very significant violation of autotolerance greatly facilitated, as is evident, for example, from the results presented here in Example 2, where immunization with wild-type Ar is compared with immunization with a variant AD molecule. It is shown (U Vat I!ei ai., 1996, 9. Ittypoi. 157: 4796-4804) that potentially autoimmune B-lymphocytes, which recognize their own proteins, are physiologically present in normal individuals. However, for the induction of these B-lymphocytes for the actual production of antibodies that react with the corresponding own proteins, the assistance of T-helper lymphocytes (Tn-cells, or Tn-lymphocytes) that produce cytokines is necessary. Usually, such help is not provided, because T-lymphocytes, as a rule, do not recognize T-cell epitopes derived from their own proteins when presented by antigen-presenting cells (APCs). However, if you provide a "foreign" element in your own protein (that is, introduce an immunologically significant modification), T-cells that recognize a foreign element are activated upon recognition of a foreign epitope on APC (primarily, such as a mononuclear cell). Polyclonal B-lymphocytes (which are also APCs) capable of recognizing self-epitopes on modified self-proteins also internalize antigen and subsequently present foreign) T-epitope(s) from it, and activated T-lymphocytes to such autoimmune polyclonal B-lymphocytes subsequently provide help with cytokines. Since the antibodies produced by these polyclonal B-lymphocytes react with different epitopes on the modified polypeptide, including those also present on the native polypeptide, an antibody is induced that cross-reacts with the unmodified self protein. In conclusion, T lymphocytes can be activated as if a population of polyclonal B lymphocytes recognized a completely foreign antigen, when in fact only the integrated epitope(s) are foreign to the host. Thus, antibodies capable of cross-reaction with unmodified self-antigens are induced.

In this area, several ways of modifying the peptide's own antigen are known to achieve a violation of autotolerance. But it is still preferable that the analog according to this invention includes - at least one first introduced group that targets the modified molecule to the antigen-presenting cell (APC) and/or - at least one second introduced group that stimulates the immune system , and/or - at least one third introduced group that optimizes the presentation of the analog to the immune system.

However, all these modifications must be carried out while a significant part of the output is stored in APP or AV

B-cell epitopes, since the recognition of the native molecule by B-lymphocytes is thus enhanced.

In one of the preferred implementations, covalently or non-covalently introduced side groups (in the form of foreign T-cell epitopes or the above first, second and third groups). This means that branches from amino acid residues obtained from APP or Ar are derivatized without changing the primary amino acid sequence or at least without making changes in the peptide bonds between individual amino acids in the chain.

In an alternative and preferred implementation, amino acid replacement and/or deletion, and/or insertion, and/or addition are used (which can be carried out using recombinant methods or by peptide synthesis; modifications involving longer stretches of amino acids can lead to formation of hybrid polypeptides). One of the particularly preferred versions of this implementation is the technology described in M/O 95/058491|, which describes a method of down-regulation of own proteins by immunization with analogs of own proteins, where a section of the amino acid sequence (a series of sequences) is replaced by a corresponding section of the amino acid sequence(s) , each of which contains a foreign immunodominant T-cell epitope, while at the same time the analog retains the complete tertiary structure of its own protein. However, for the purposes of this invention, it is essential if the modification (whether it is an insertion, addition, deletion or replacement) leads to the formation of a foreign T-cell epitope and simultaneously preserves a significant number of B-cell epitopes in APP or AD. However, in order to obtain the maximum efficiency in the induction of an immune response, it is preferable that the complete tertiary structure of APP or AB is preserved in the modified molecule.

In some cases, it is preferable that APP or Ar, or their fragments, be mutant. Substitution variants where methionine at position 35 in Ar-43 is replaced, preferably by lysine or isoleucine or simply deleted, are especially preferred. Particularly preferred analogues contain a single methionine located at the C-terminus, either because it occurs naturally in the amyloidogenic polypeptide or foreign Tn epitope, or because it has been inserted or added there. Therefore, it is also preferred that the region of the analog that includes the Tn epitope also does not contain methionine, excluding the possible C-terminal location of methionine.

The main reason for removing all but one of the methionines is that it becomes possible to recombinantly obtain polymeric analogs that can be sequentially cleaved with cyanide bromide to yield a single analog. The advantage is that recombinant products facilitate this way.

In fact, it is generally preferred that all APP or Aβ analogs used in accordance with the present invention possess the characteristic feature of simply incorporating a single methionine located in the analog as the C-terminal amino acid, and that all other methionines either in the amyloidogenic polypeptide, or in a foreign Tn-epitope were deleted or replaced by another amino acid.

An additional interesting mutation is a deletion or substitution of phenylalanine at position 19 of Ar-43, and it is particularly preferred that the mutation is a replacement of a phenylalanine residue with a proline.

Other interesting polyamino acids for use in analogs are truncated parts of the protein Ar-43. They can be used in immunogenic analogs according to this invention. Shortened parts of AD (1-42), AD (1-40), AD (1-39), AD (1-35), AD (1-34), Ar (1-28), Ar (1 -12), A (1-5), AD (13-28), AD (13-35), Ar (17-28), AB (25-35), Ar (35-40), Ar (36- 42) and Ar (35-42) (where the numbers in parentheses mean segments of amino acids Ar-43, which make up the corresponding fragment: for example, Ar (35-40) is identical to amino acids 706-711 in BEO IU MO:2). All these variants with shortened parts of Ar-43 can be obtained with the AD fragments described here, specifically with variants 9, 10, 11, 12, and 13, indicated in example 1.

The following formula describes the molecular structures related to the invention: (MOOB))5i(amyloidet)n(MYU»)zg(amyloideg) po... () (МОВх»х)«hamyloidech) х where amyloides - amyloidech represent x A B-cell epitope containing APP or Aβ subsequences that are independently identical or non-identical and that may contain foreign side groups, x is the number 23, pi-x are the x numbers 20 (at least one x1), MO0BI- MY00x represent x modifications introduced between the B-cell epitopes presented, and 51-5x represent x numbers of 20 (at least one»1!, if no side group is introduced in the amyloid sequence). Thus, taking into account the general functional restrictions on the immunogenicity of the design, the invention allows all kinds of permutations of the original sequence of APP or Ar and all kinds of modifications in it. Thus, the invention includes modified APP or AD obtained by omitting parts of the sequence, which, for example, reveal ip without adverse effects, or omitting parts that are usually intracellular and, thus, can give rise to unwanted immunological reactions.

One preferred version of the designs outlined above, when applicable, is a design where the subsequence containing the B-cell epitope of the amyloid protein is not exposed extracellularly in the precursor polypeptide from which the amyloid is derived. This selection of epitopes ensures that no antibodies are produced that could react with the progenitor-producing cells, and thus the immune response produced becomes a narrowly directed immune response against unwanted amyloid deposits.

For example, in this case, it is possible to induce immunity against APP or AD epitopes, exposed in the extracellular phase, only when they are free from any connection with the cells that produce them.

Retention of a substantial portion of the B-cell epitopes or even the entire tertiary structure of the protein to be modified, as desired here, can be achieved in several ways. One of them is the simple preparation of a polyclonal antiserum directed against the polypeptide under consideration (for example, an antiserum obtained from a rabbit), and the use of this antiserum subsequently as a test reagent (for example, competitive EGI5A) against the obtained modified proteins. Modified versions (analogs) that react with antiserum in the same volume as APP or AD must be considered as having exactly the same tertiary structure as APP or AD, while analogs that show limited (but all still significant and specific) reactivity with such an antiserum is considered as having preserved a significant part of the original B-cell epitopes.

Alternatively, it is possible to select monoclonal antibodies reacting with different epitopes on

APP or AD, and use them as a test panel. This approach has the advantage of allowing 1) mapping of APP or Aβ epitopes and 2) mapping of epitopes conserved in the resulting analogs.

Of course, in the third approximation one should determine the 3-dimensional structure of APP or AD, or their biologically active shortened product (cf. above) and compare them with the determined 3-dimensional structure of the obtained analogues. The three-dimensional structure can be distinguished by studying X-ray diffraction and NMR spectroscopy. Additional information about the tertiary structure can be obtained to some extent by studying circular dichroism, which has the advantage that the only thing necessary to obtain useful information about the tertiary structure of a given molecule is a purified polypeptide (whereas in X-ray diffraction it is necessary to provide a crystallized polypeptide, and for NMR it is necessary to provide isotopic variants of the polypeptide). However, in the end X-ray diffraction and

NMR is necessary to obtain convincing data, since circular dichroism can only provide indirect evidence of the correctness of the 3-dimensional structure by means of information about the elements of the secondary structure.

In one of the preferred implementations of the invention, multiple representations of B-lymphocyte epitopes of APP or Ar are used (for example, the formula | where at least one B-cell epitope is represented in two positions). This effect can be achieved in various ways, for example, by simply preparing hybrid polypeptides containing the structure (APP-derived or AD polypeptide)t, where t is the number 22, and then introducing the modifications discussed herein into at least one of the APP sequences or

HELL. Preferably, the modifications that are introduced include at least one duplication of the B-lymphocyte epitope and/or the introduction of a hapten. Embodiments including multiple representations of selected epitopes are particularly preferred in situations where only small portions are suitable as components of vaccine formulations.

APP or AD.

As indicated above, the introduction of a foreign T-cell epitope can be achieved by inserting, adding, deleting or replacing at least one amino acid. Of course, in the usual situation more than one change in amino acid sequence is introduced (eg, insertion or replacement of an entire T-cell epitope), but an important goal to achieve is that the antigen-presenting cell (ARC) analog will give rise to to such a foreign immunodominant T-cell epitope presented in connection with the MHC class II molecule on the surface of ARS. Thus, if the APP or Aβ amino acid sequence at the appropriate positions contains a number of amino acid residues that can be detected in a foreign Tn epitope, then introduction of a foreign Tn epitope can be achieved by providing the remaining amino acids to the foreign epitope by insertion, addition, deletions or substitutions of amino acids. In other words, introduction of the entire Tn epitope by insertion or replacement is not necessary to fulfill the purposes of the present invention.

Preferably, the number of amino acid insertions, deletions, substitutions or additions is at least 2, for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, and 25 insertions, substitutions, additions, or deletions. In addition, it is preferable that the number of insertions, substitutions, additions, or deletions of amino acids does not exceed 150, for example, no more than 100, no more than 90, no more than 80, and no more than 70. It is especially preferable that the number of substitutions, insertions, deletions, or additions does not exceed 60, and in particular, this number should not exceed 50 or even 40. Most preferably, the number does not exceed 30. In relation to amino acid additions, it should be noted that when the resulting construct is in the form of a hybrid polypeptide, there are often significantly more than 150.

Preferred implementations of this invention include modification by introducing at least one foreign immunodominant T-cell epitope. It should be understood that the question of immunodominance of a T-cell epitope depends on the species of animal under consideration. Here, the term "Immunodominance" literally refers to epitopes that elicit a significant immune response in vaccinated individuals/populations, but it is a well-known fact that a T-cell epitope that is immunodominant in one individual/population is not necessarily immunodominant in another individual of the same species, even despite the fact that it is capable of binding to INO-HER molecules in the last individual. Hence, for the purposes of this invention, an immunodominant T-cell epitope is a T-cell epitope that, when presented in an antigen, will effectively elicit T-cell help. Usually, immunodominant T-cell epitopes have such a natural ability that they are essentially always presented bound to MN class II molecules, regardless of the polypeptide in which they are found.

Another important issue is the problem of MHC restriction of T-cell epitopes. As a rule, natural T-cell epitopes are MHC-restricted, that is, certain peptides that make up the T-cell epitope will effectively bind only to a subset of MHC class II molecules. This in turn has the effect that, in most cases, the use of one specific T-cell epitope will result in a vaccine component that is only effective for a subset of the population, and depending on the size of that subset, it may be necessary to include more T-cell epitopes into the same molecule or, alternatively, to prepare a multicomponent vaccine, where the components are variants of APP or AD, different from each other by the nature of the introduced T-cell epitope.

If the MNeO-restriction of the T cells to be used is unknown (for example, in a situation where the animal being vaccinated has an ill-defined MHC composition), the fraction of the population covered by a particular vaccine composition can be approximated using the following formula: n

Ipopul -1- PO - ri) (1) i-1 where ri represents the frequency of responders in the population to the i-th foreign T-cell epitope presented in the vaccine composition, and n represents the total number of foreign T-cell epithets. Thus, a vaccine composition containing C foreign T-cell epitopes that have population response frequencies of 0.8, 0.7, and 0.6, respectively, should give

1-0.2x0.3x0.4-0.976, that is, 97.6 percent of the population will statistically increase the mediated INO-1I response to the vaccine.

The formula above is not used in situations where the MNO-restriction profile of the applied peptides is more or less precisely known. If, for example, the determined peptide binds only to human MNO-1 molecules, encoded by alleles of ХОКИ, ХОКЗ, ОМ5 and ОК7 NHA-OK, then the use of this peptide together with another peptide that binds to МНО-ХІ molecules, remaining ones encoded by NIG A-OB alleles would complete 10095 coverage in the population under consideration. Similarly, if the second peptide binds only to OZ and OK5, the addition of this peptide will not increase coverage at all. If the calculation of the population response is based only on MNSO-restriction of T-cell epitopes in the vaccine, the minimum part of the population covered by a specific vaccine composition can be determined using the following formula:

Z hypopol-1-Pr (t) - where Phi is the sum of the frequencies of allelic haplotypes encoded by MHC molecules that bind any of the T-cell epitopes in the vaccine and belong to the th of the known HA loci ( OR, OK and 0О) in the population; in practice, it is first determined which MHC molecules recognize each T-cell epitope in the vaccine, and then they are sorted by type (OR, OK and 09); then, for each type, individual frequencies of various sorted allelic haplotypes are summed up, thus obtaining (Fi, fg and Fz.

It may happen that the value of p; in formula II will exceed the corresponding theoretical value of li

Z t -1- PM (M) i-th where y; represents the sum of allelic haplotype frequencies encoded by MH molecules that bind the i-th

T-cell epitope in the vaccine and belong to the )th of the known NHA loci (OR, OK and BO) in the population. This means that in the 1st population, the frequency of respondents is Izalyshkova I:(ri-li/(1-li). Thus, the formula of ЯЙ can be transformed in such a way as to obtain the formula M:

With p

Ipopul -1- by -FiYi tsh1- by -Valyshov 1) (M) i-i i-i where the term Bbalyshkov ii is set to zero if it is negative. It should be noted that formula M requires that all epitopes map to identical sets of haplotypes.

Therefore, when choosing T-cell epitopes for introduction into the analogue, it is important to take into account all available information about epitopes: 1) the frequency of responders in the population for each epitope, 2) data on INR-restriction and 3) the frequency of the corresponding haplotypes in the population.

There are a number of natural "mixed" T-cell epitopes active in a large proportion of individuals of an animal species or animal population, and these are preferably introduced into a vaccine, thus reducing the need for a large number of different analogues in a single vaccine.

The mixed epitope according to the invention can be a natural human T-cell epitope, such as epitopes from tetanus toxoid (for example, epitopes P2 and RZO), diphtheria toxoid, hemagglutinin of influenza virus (HA) and C5-antigen of R. GaIsiragat.

Over the years, a number of other mixed T-cell epitopes have been identified. Mainly identified peptides capable of binding to a large part of NI A-OK molecules encoded by different NIGA-OK alleles, and they are all T-cell epitopes admissible for introduction into analogues used in accordance with this invention. Cf. also the epitopes discussed in the following references, which are thus all incorporated herein by reference: (MO 98/23635 (Rgaeg IN. ei ai., submitted to Tpe Opimeg5yu oi Oceepziapa); Bosch pmooa 5. ei. a !., 1998, 9. Ittipoi. 160: 3363-3373; bipidadiia R. ei ai., 1988, Maishige 336: 778-780; Spisl: V.M. ei a!., 1993, 9. Ehr. Mei. 178: 27-47; Natteg" .. ei ai., 1993, Seii 74: 197-203; apa haik K. ei ai., 1994, Ittipodepeiiisv 39: 230-242). The last reference also refers to ligands NGA-0O and A. All of the epitopes listed in these 5 references are suitable as natural candidate epitopes for use in accordance with the present invention as epitopes sharing common motifs with them.

Alternatively, the epitope may be any artificial T-cell epitope capable of binding to a large portion of class II MHC molecules. In this context, peptides that are common epitopes for all OCs ("RAOKEs") are described in (M/O 95/07707 and in the corresponding article by Alekhapadeg U. eyi a!., 1994, Ittipku 1: 751-761 (both descriptions incorporated herein by reference)|, are interesting candidates for epitopes for use in accordance with the present invention. It should be noted that the most effective RAOKE peptides described herein carry Y-amino acids at the C- and M-termini to increase stability upon administration However, the present invention primarily seeks to introduce the relevant epitopes as part of an analog that is subsequently enzymatically degraded within the lysosomal compartment

APC to allow further presentation in connection with the molecule MNO-1II, and therefore, the introduction of 10-amino acids in the epitopes used according to the invention is inappropriate.

One of the particularly preferred RAOKE peptides is a peptide having the amino acid sequence AKEMAAUMTICAAA (5EO IO MO:17) or its immunologically effective subsequence. This and other epitopes with the same lack of MHC restriction are preferred T-cell epitopes that must be present in analogues used in the method according to the invention. Such a supermixed epitope is acceptable for most simple implementations of the invention, where only one analog is presented to the vaccinated animal's immune system.

As indicated above, the modification of APP or AD may also include the introduction of the first group targeting the modified amyloidogenic polypeptide to the APC or B-lymphocyte. For example, the first group can be a specific binding partner for a specific surface antigen of a B-lymphocyte or for a specific surface antigen of APC. Many such surface-specific antigens are known in this field. For example, the group can be a carbohydrate for which there is a receptor on a B-lymphocyte or

ARS (eg, mannan or mannose). Alternatively, the second group may be a hapten. Also, as the first group, you can use an antibody fragment that specifically recognizes the surface molecule on

APC or lymphocytes (for example, the surface molecule may be a macrophage or monocyte ECu receptor, such as Esuki, or, alternatively, any other surface-specific marker, such as

CO40 or STI A-4). It should be noted that all these exemplified molecules can also be used as part of an adjuvant, the comparison is below.

As an alternative or in addition to targeting the analog to a specific cell type to achieve an enhanced immune response, the level of reactivity of the immune system can be increased by including the second immune-stimulating group indicated above. Typical examples of such second groups are cytokines and heat shock proteins or chaperone molecules, as well as their effective parts.

Cytokines suitable for use according to this invention are cytokines that will also function normally in the vaccine composition as adjuvants, i.e., for example, interferon y (IEM-y), interleukin 1 (I--1), interleukin 2 (I- -2), interleukin 4 (1--4), interleukin 6 (1--6), interleukin 12 (1-12), interleukin 13 (I--13), interleukin 15 (I--15) and granulocyte- macrophage colony-stimulating factor (CM-С5РЕ); alternatively, as a second group, the functional part of the cytokine molecule may be sufficient. For the use of such cytokines as adjuvant agents, compare the discussion below.

Suitable heat shock proteins or molecular chaperones used as a second group according to this invention can be НОР7О, Н5РО0, НСТО, SEROA | also known as 9р9ob, cf. Uueagzsp RA eyi a. 1998, Viospetivighu 37: 5709-19) and SKT (calreticulin).

Alternatively, the second group can be a toxin such as listeriolysin (GO), lipid A, and heat-labile enterotoxin. Interesting possibilities are also associated with mycobacterial derivatives such as MOR (muramyldipeptide), CEPA (complete Freund's adjuvant) and trehalose esters TOM and TOB.

An important implementation of this invention is also the possibility of introducing a third group that enhances the presentation of the analog to the immune system. Several examples of this principle are shown in this area. For example, it is known that the palmitic acid lipoylation anchor in the O5pA protein in Vogtiiia riggegi can be used to provide polypeptides that are themselves adjuvants (cf., for example, UUO 96/40718) - it appears that lipoylated proteins form structures , similar to micelles, with a core consisting of parts of the lipopolypeptide anchors, and the remaining parts of the molecule protruding from there, lead to multiple presentation of antigenic determinants. Therefore, the use of such similar approaches associated with the use of different lipoylation anchors (for example, a myristyl group, a farnesyl group, a geranyl-geranyl group, an ORiI anchor, and an M-acyldiglyceride group) is a preferred implementation of this invention, since, as a rule, , the provision of such lipoylation anchors in the protein obtained by the recombinant method is quite simple and only requires, in the case of an analogue, the use of, for example, a natural signal sequence as a fusion partner. Another possibility is the use of the Cza fragment of the SZ complement factor or the SZ factor itself (cf. Oetrzeu ei ai., 1996, 5sieepse 271, 348-350 apa I osi 5 Kopieg, 1998, Mashte Vioyesppoiodu 16, 458-462.

An alternative implementation of the invention, which also leads to the preferential presentation to the immune system of multiple (for example, at least 2) copies of important sections of the APP or AD with epitopes, is the covalent binding of the analog to certain molecules, that is, options 4 and there are, indicated above. For example, polymers, such as carbohydrates such as dextrin, can be used (cf., e.g., I ee5 A ei ai., 1994, Massipe 12: 1160-1166; I eez A ei ai., 1990, 9 Ittypoi. 145: 3594-3600), but mannose and mannan are also suitable alternatives. Integral membrane proteins, such as those from E. soya and other bacteria, are also suitable conjugation partners. Traditional carrier molecules such as sea urchin hemocyanin (CIN), tetanus toxoid, diphtheria toxoid, and bovine serum albumin (B5A) are also preferred and suitable for conjugation partners.

Preferred implementations of covalently linking an APP- or AD-derived substance to polyhydroxy polymers such as carbohydrates include the use of at least one APP- or Aβ-derived peptide and at least one foreign T-helper epitope that separately bind to polyhydroxypolymer (that is, the foreign T-helper epitope and the amino acid sequence derived from APP or AD do not fuse with each other, but largely bind to the polyhydroxypolymer, which serves as a supporting framework). On the other hand, the most preferred implementation is when the corresponding regions of the APP-derived or Aβ-derived peptides carrying the B-cell epitope are assembled using short peptide segments, because this approach represents a very convenient way to achieve multiple presentation of selected epitopes in final immunogen. However, it is also possible to simply link the analogues already described here to the polyhydroxypolymeric backbone, i.e. so that the substance obtained from

APP or AD did not connect to the skeleton separately from foreign Tn-epitopes.

It is especially preferred that the binding of the foreign T-helper epitope and the APP or Aβ-derived polypeptide occurs via an amide bond that can be cleaved by peptidase. This strategy has the effect that APCs will be able to capture the conjugate and subsequently present of a foreign T-cell epitope in connection with MNS class II.

One of the ways to achieve the binding of peptides (and APP- or Ar-derived peptides of interest, as well as foreign epitope) is the activation of the corresponding polyhydroxy polymer with tresyl (trifluoroethylsulfonyl) groups or other appropriate activating groups,

such as maleimide, para-nitrophenylchloroform (for activation of OH groups and formation of a peptide bond between the peptide and polyhydroxypolymer) and tosyl (para-toluenesulfonyl). For example, activated polysaccharides can be prepared as described in (MUO 00/05316 and 05 5874469 (both incorporated herein by reference))| and link them with peptides or polyamino acids obtained from APP or AD, as well as with T-cell epitopes obtained using standard methods of peptide synthesis in the liquid or solid phase.

The resulting product consists of a polyhydroxypolymer backbone (for example, a dextran backbone), which is linked by its M-ends or other nitrogen groups to polyamino acids derived from APP or Ar and from foreign T-cell epitopes. If desired, APP or AD peptides can be synthesized to protect all available amino groups except for one at the M-terminus, subsequently linking the resulting protected peptides to tresylated dextran groups and finally deprotecting the resulting conjugate. A specific example of this approach is described in the examples below.

Instead of using water-soluble polysaccharide molecules, as described in MUMO 00/05316 and 05 5874469), it is equally possible to use cross-linked polysaccharide molecules, thus obtaining corpuscular conjugates between polypeptides and polysaccharides, which is believed to lead to improved presentation of polypeptides to the immune system, since two goals are achieved, namely: obtaining the effect of local accumulation upon injection of the conjugate and obtaining particles that are more attractive targets for ARS. This approach using such systems is also discussed in detail in the examples.

The considerations underlying the selection of areas for introducing modifications in APP or AD include a) preservation of known and predicted B-cell epitopes, B) preservation of the tertiary structure, c) avoidance of presentation of B-cell epitopes on "producer cells" etc. At least as discussed above, screening a set of analogues, all of which have been subjected to T-cell epitope insertion at different positions, is quite straightforward.

Since the most preferred embodiments of the present invention involve the down-regulation of human Aβ, it is therefore preferred that the APP or AD polypeptide discussed above is a

AD of a person. In this implementation, it is particularly preferred that the APP or AD polypeptides are modified by replacing at least one amino acid sequence in ЖЭО ЮЮ MO:2, one amino acid sequence of equivalent or different length containing a foreign Tn epitope. Preferred examples of modified amyloidogenic APP and AR are schematically shown in figure 1, using epitopes P2 and RZO as examples. The logical justification of such constructions is discussed in detail in the examples.

More specifically, the amino acid sequence containing Tn, which is introduced into the 5EO IU MO:2, can be introduced from any amino acid into the ZEO IU MO:2. That is, introduction is possible after any of the amino acids in positions 1-770, but preferably after any of the amino acids in positions 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683 , 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 707 , 709, 710, 711, 712, 713 and 714 in ZEO IO MO:2. This can be combined with deletion of any or all of the amino acids at positions 1-671 or any or all of the amino acids at positions 715-770. In addition, when using the method of replacing any of the amino acids in positions 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713 and 714 in ZEO IU MO:2 can be deleted together with this entry.

Another implementation of this invention is related to the presentation of analogs that do not contain any of the subsequences of 5EO IU MO:2, which productively bind to MHC molecules of class II, initiating a T-cell response.

The rationale for such a strategy to construct an immunogen that recruits the immune system to induce, for example, an immune response against Aβ, is as follows: it has been reported that upon immunization with multiple autologous proteins, such as AD, adjuvanted, that are strong enough to impair tolerance of the body to autologous proteins, there is a danger that in the same vaccinated individuals the induced immune response will not be able to stop simply due to the cessation of immunization. This is because the induced immune response in such individuals is most likely to trigger the native Tn epitope of the autologous protein, and this has the adverse effect that the vaccinated individual's own protein will be able to function as an immunogen: thus, an autoimmune state will be established.

In the preferred methods, including the use of foreign Tn-epitopes, which are better to the knowledge of the authors of the present invention, this effect has never been observed, since the immune response against the "self" is directed by the foreign Tn-epitope, and the authors of the present invention have repeatedly shown that the induced immune the response induced by the use of the predominant technique did decrease after cessation of immunization. However, in theory it may happen in a few individuals that the immune response will also be directed by an autologous Tn epitope of the corresponding self protein immunized against, which is particularly relevant when considering self proteins that are relatively abundant, such as AD, while others are therapeutically significant own proteins are present in the body only locally or in such small quantities that the "self-immunization effect" is impossible. One very simple way to avoid this is, therefore, to completely avoid including in the immunogen peptide sequences that can serve as Tn epitopes (and since peptides shorter than about 9 amino acids cannot serve as Tn epitopes, using shorter fragments is one simple and acceptable approaches). Therefore, this embodiment of the invention also serves to ensure that the immunogen does not include peptide sequences of target APPs or Ars that may serve as "self-stimulatory Tn epitopes," including sequences that simply contain conservative substitutions in the target protein sequence. which may otherwise function as a Tn epitope.

Preferred implementations of presentation to the immune system of analogues of APP or AD include the use of a chimeric peptide that contains at least one peptide derived from APP or AD, which does not productively bind to MHC class II molecules, and at least one foreign T-helper epitope. Moreover, it is preferable that the peptide obtained from APP or AD carries a B-cell epitope. It is especially preferred if the immunogenic analog is an analog where the amino acid sequences include one or more B-cell epitopes, presented either as a continuous sequence or as a sequence containing inserts, where the inserts include foreign T-helper epitopes.

On the other hand, the most preferred implementation is when the corresponding sections are derived from peptides

APP or AD, which carry the B-cell epitope, are composed of short peptide segments that are not able to productively connect with the MHC class II molecule. The selected B-cell epitope or epitopes of the amyloidogenic polypeptide must therefore include no more than 9 consecutive amino acids of ZEO IU

MO:2. Shorter peptides are preferred, such as peptides having no more than 8, 7, 6, 5, 4 or 3 consecutive amino acids of the amino acid sequence of the amyloidogenic polypeptide.

Preferably, the analogue includes at least one subsequence of ZEO IU MO:2, so that each of these at least one subsequences independently consists of amino acid segments of APP or AD selected from the group consisting of 9 consecutive amino acids, 8 consecutive amino acids, 7 consecutive amino acids, 6 consecutive amino acids, 5 consecutive amino acids, 4 consecutive amino acids and 3 consecutive amino acids.

It is particularly preferred that consecutive amino acids begin with an amino acid residue selected from the group consisting of residues 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688 . BEO AND MOS2.

Protein/peptide vaccination; drugs and introduction of analogues

When the analog is presented to the animal's immune system by administering it to the animal, the polypeptide preparations meet the requirements generally accepted in the field.

The production of vaccines containing as active ingredients peptide sequences is generally well established in the art, as illustrated (US Patents 4,608,251, 4,601,903, 4,599,231, 4,599,230, 4,596,792, and 4,578,770, all of which are incorporated herein by reference). Usually, such vaccines are obtained as preparations for injections or as liquid solutions or suspensions; it is also possible to obtain solid forms suitable for dissolution or suspension in a liquid before injection. Preparations can also be emulsified. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of excipients such as wetting agents or emulsifiers, buffering agents for pH stabilization, or adjuvants that increase the effectiveness of vaccines; cf. a detailed discussion of adjuvants below.

Usually, vaccines are administered parenterally, by injection, for example, or subcutaneously, or intravenously, or intramuscularly. Additional preparations suitable for other routes of administration include suppositories and, in some cases, oral, buccal, sublingual, intraperitoneal, intravaginal, anal, epidural, spinal and intracranial preparations. For suppositories, standard binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range from 0.595 to 1095, preferably - 1-295. Oral preparations include such commonly used fillers as, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharinate, cellulose, pharmaceutical grade magnesium carbonate, etc. These compositions take the form of solutions, suspensions, tablets, pills, capsules, drugs with delayed release or powders and contain 10-9595 active ingredient, preferably - 20-7095. For oral preparations, a suitable formulation component (and also a possible conjugation component) is cholera toxin.

Polypeptides can be formed into vaccines in the form of neutral forms or in the form of salts.

Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the peptide) and those salts formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or with such organic salts as acetic, oxalic, tartaric, mandelic. etc. Salts formed with free carboxyl groups can also be prepared with inorganic bases, for example, such as sodium, potassium, ammonium, calcium or iron hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. .p.

Vaccines are administered in a manner compatible with drug dosing and in an amount that is therapeutically effective and immunogenic. The amount to be administered depends on the subject to be treated, including, for example, the ability of the individual's immune system to mount an immune response and the degree of protection required. Suitable dosage ranges are within the range of about several hundred micrograms of active ingredient per vaccination, with a preferred range of from about 0.1 µg to 2000 µg (although larger amounts in the range of 1-10 mg are also contemplated), for example in the range of about 0.5 µg to 100 µg, preferably in the range of 10 µg to 50 µg and, especially, in the range of about 10 µg to 10 µg.

Appropriate regimens for initial administration and maintenance doses also vary, but are defined by initial administration followed by vaccination or other forms of administration.

The method of application can vary widely. Any of the traditional methods of vaccine administration is applicable. They include oral use on a solid physiologically acceptable basis or in a physiologically acceptable dispersion, parenterally, by injection, etc. The dosage of the vaccine will depend on the method of administration and will vary depending on the age of the subject to be vaccinated and the composition of the antigen.

Some of the vaccine polypeptides are quite immunogenic in the vaccine, but for some others the immune response will be enhanced if the vaccine additionally contains an activator.

Different methods of achieving an activating effect are known for vaccines. The basic principles and methods are well developed in | TPne Tpeogu apa Rgasiisa! Arrisaiop oi Adiimapiv", 1995, YuOipsap E.5. 5iezhapy-Tii (ea.), add.

MMieu ope TSO, IZVM 0-471-95170-6, and also in "Massipe5: Mem/ Sepegaiopp Ittypoiodisa! Adiimapiv", 1995,

Stedogiadie S eyi ai. (ed5.), Riepyt Rhev5, Mem Mkhoiik, IZVM 0-306-45283-9, included here by reference).

It is especially preferable to use an adjuvant, which, as can be shown, contributes to the violation of autotolerance to autoantigens; in fact, it is significant in cases where unmodified amyloidogenic polypeptide is used as an active ingredient in the autovaccine. As non-limiting examples, suitable adjuvants are selected from the group consisting of an immune-targeting adjuvant; an immunomodulatory adjuvant, such as a toxin, cytokine and derivatives of mycobacteria; oil composition; polymer; adjuvant that forms micelles; saponin; immunostimulating complex matrix (IZSOM matrix); particles; WASP; adjuvants based on aluminum; DNA-based adjuvants; y-inulin and encapsulating adjuvant. In general terms, it should be noted that the descriptions above, which referred to compounds and means suitable as the first, second and third groups in analogues, with appropriate changes, also refer to their use in a vaccine adjuvant according to the invention.

The possible use of adjuvants includes the use of such agents as aluminum hydroxide or phosphate (alum), which is usually used as a 0.05-0.1 percent solution in a buffered saline solution, an admixture with synthetic polymers of sugars (for example, Sagroro!F ), applied as a 0.25 percent solution, aggregation of proteins in the vaccine by exposure to heat with a temperature range from 70 to 101"C for a period of 30 seconds to 2 minutes, respectively, and aggregation using structuring substances. Also albumin aggregation by reactivation of pepsin-treated antibodies (Bar-fragments), admixture with bacterial cells such as C raglite, or endotoxins, or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil carriers such as manid monooleate (Agassey A) or an emulsion with a 20 percent solution of perfluorocarbon (Riyo50I-YUSA) used as a blocking substitute.A mixture with oils such as squalene and IRA is also preferred.

According to the present invention, OBA (dimethyldioctadecylammonium bromide) is an interesting adjuvant candidate, as are DNA and γ-inulin, but complete and incomplete Freund's adjuvants, as well as chyla saponins such as

OMA ii 0521, also interesting, as well as KIVI. Additional options include monophospholipid A (MP), listed above

CVD and Cz3a, and muramyldipeptide (MOP).

Liposome preparations are known to have adjuvant effects, and therefore, adjuvants in the form of liposomes are preferred according to the invention.

Preferred alternatives according to the invention are also adjuvants in the form of an immunostimulating complex matrix (IESOMF matrix)), especially because it has been shown that this type of adjuvant is capable of up-regulating the expression of MHC class I of ARS. The matrix of IZSOMF consists of (optionally fractionated) saponins (triterpenoids) from Ocia)a zaropagia, cholesterol and phospholipid. When this is mixed with an immunogenic protein, the resulting particulate preparation is what is known as an IZCOM particle, where the saponin is 60-7095 w/w, the cholesterol and phospholipid are 10-1595 w/w, and the protein is 10 -1595 wt./wt. Details related to the composition and use of immunostimulating complexes can be found, for example, in the above-mentioned textbooks devoted to adjuvants, but in (Mogeip Vei ai., 1995, Siip. IttypoiNeg. 3: 461-475, as well as in Vagtg IS apa Miispeii SE, 1996, Ittipoi, apa Sei!Visi.74: 8-25 (incorporated herein by reference)|also provide suitable instructions for preparing complete immunostimulatory complexes.

Another very interesting (and thus preferred) possibility of achieving an adjuvant effect is associated with the use of the method described in |Sozzeiip et al., 1992 (which is, therefore, incorporated herein by reference)). Briefly, presentation of an appropriate antigen, such as an antigen according to the present invention, can be enhanced by conjugation of the antigen with antibodies (or antigen-binding fragments of antibodies) against Esu receptors on monocytes/macrophages. It is shown that immunogenicity for vaccination purposes is especially enhanced by conjugates between antigen and anti-EsuVi.

Other possibilities include the use of targeting and immunomodulatory agents (eg, cytokines) listed above as candidates for the first and second groups in modified versions of amyloidogenic polypeptides. In this regard, synthetic inducers of cytokines, such as poly I:C, are also possibilities.

Suitable mycobacterial derivatives are selected from the group consisting of muramyldipeptide, complete adjuvant

Freund, Kiwi and complex diesters of trehalose, such as TOM or TOE.

Suitable immune-targeting adjuvants are selected from the group consisting of CO40 ligand and anti-CO40 antibodies or specifically binding fragments thereof (see discussion above), mannose, Rar fragment, and STI A-4.

Suitable polymeric adjuvants are selected from the group consisting of carbohydrates such as dextran, RES, starch, mannan and mannose; plastic polymers and latex such as latex granules.

Another interesting way of modulating the immune response is the inclusion of an immunogen (optionally together with adjuvants and pharmaceutically acceptable carriers) in a "virtual lymph node" (MI M) (patented medical device developed in IttipoTPegar, Ips., 360 I ehipdiop Amepie , Mem/ MogkK, MU 10017-6501). MM (thin tubular device) mimics the structure and function of a lymph node. Entry

MM under the skin creates an area of sterile inflammation with increased levels of cytokines and chemokines. T- and B-cells, as well as ARS, quickly respond to damage signals, are directed to the site of inflammation and accumulate inside the porous matrix of MM. It has been shown that the required dose of antigen required to establish an immune response to the antigen with the use of MIM is reduced and that the immunoprotection caused by immunization with the use of MIM exceeds standard immunization with the use of KIVI as an adjuvant. The technology is briefly described in |Seireg Seyi ai!., 1998, "Eisyainop oi Noriva

Seyishag apa Nitoga! Ittype NKezropze5 Yu Ztai! Atoshpiv oi Ittipodepe Ovipd a Mome! Meadaisa! Ohemise

Oezidpageid te mipshai! utry Mode", ip: "Egot (pe I arogaigu o She Siipis, Vook oi Arvigasiv, Osioreg 1217-1517 1998, beazsare Vezoii, Ariov, Sai Pogpia"|.

It is shown that vaccine preparations in the form of microparticles in many cases increase the immunogenicity of protein antigens, and therefore represent another preferred embodiment of the invention. Microparticles are made either as compositions together with a polymer, lipid, carbohydrate or other molecules suitable for the manufacture of particles, or microparticles can be homogeneous particles consisting only of the antigen itself.

Examples of microparticles based on polymers are particles based on RI CA and

RMR ISoiria, K. K. eyi. AI. 1998), where the polymer and antigen condense into a solid particle. Lipid-based particles can be produced as lipid micelles (so-called liposomes) by capturing the antigen in the micelle |Rieigorop, R.

U. 1995). Carbohydrate-based particles are usually made from a suitable degradable carbohydrate, such as starch or chitosan. Carbohydrate and antigen are mixed and condensed into particles in a process similar to that used for polymer particles | Kav, N. 5. ei ai. 1997).

Particles consisting only of antigen can be produced by various methods of spraying or freeze-drying. For the purposes of this invention, the supercritical fluid method, which is used for the production of very uniform particles of controlled size, is particularly suitable | Mogk, R. 1999 5

Zpekypom, V. ei a. 1999).

The vaccine is expected to be administered 1-6 times a year, ie 1, 2, 3, 4, 5 or 6 times a year, to an individual who needs it. It has previously been shown that the immunological memory induced by the use of the preferred autovaccines according to this invention is not permanent, and therefore the immune system requires periodic provocation of the immune response with an amyloidogenic polypeptide or modified amyloidogenic polypeptides.

As a result of genetic diversity, different individuals can respond to the same polypeptide with an immune response of different strength. Therefore, the vaccine according to the invention may contain several different polypeptides to increase the immune response, cf. also the discussion above regarding the choice of foreign T-cell epitope insertions. The vaccine may include two or more polypeptides, where all polypeptides are as defined above.

Therefore, the vaccine can include 3-20 different modified or unmodified polypeptides, for example, 3-10 different peptides.

Nucleic acid vaccination

As an alternative to classical protein-based vaccine administration, nucleic acid vaccination technology (also known as "nucleic acid immunization," "genetic immunization," and "gene immunization") offers a number of attractive features.

First of all, in contrast to the traditional approach to vaccination, nucleic acid vaccination does not require resources that require large-scale production of the immunogen (for example, on an industrial scale fermentation of microorganisms that produce amyloidogenic polypeptides). Moreover, there is no need for purification devices and immunogen refolding schemes. Finally, since nucleic acid vaccination relies on the biochemical machinery of the individual being vaccinated for the expression product of the introduced nucleic acid, optimal post-translational processing of the expressed product is expected to occur; this is especially important in the case of autovaccination, since, as indicated above, a significant part of the original B-cell epitopes must be preserved in the modified molecule and since B-cell epitopes can in principle be composed of parts of any (biological) molecule (e.g., carbohydrate, lipid , protein, etc.). Thus, the natural glycosylation and lipolysis profiles of an immunogen can be very important for overall immunogenicity, and this is best ensured by using the host as the producer of the immunogen.

Therefore, the preferred implementation of options a-c according to the invention includes effective presentation of the analog to the immune system by introducing nucleic acid(s) encoding the analog into the cells of the animal and thus obtaining the expression of the introduced(s) of nucleic acid(s) by cells and mimo.

In this embodiment, the introduced nucleic acid is preferably DNA, which can be in the form of pure DNA, DNA combined with charged or uncharged lipids, DNA placed in liposomes, DNA included in a viral vector, DNA combined with a transfection-facilitating bileume or polypeptide, DNA fused to a targeting protein or polypeptide, DNA fused to calcium-precipitating agents, DNA linked to an inert carrier molecule, DNA encapsulated in a polymer, for example, in RI CA Compare the microencapsulation method described in M/O 98/313981I, or chitin, or chitosan and

DNA combined with an adjuvant. In this context, it should be noted that almost all the options considered, which relate to the use of adjuvants in the preparations of traditional vaccines, are also used in the preparations of DNA vaccines. Therefore, all descriptions that refer to the use of adjuvants in the context of vaccines based on polypeptides, with the necessary changes, apply to use in the method of vaccination with nucleic acids.

The routes and regimens for administration of polypeptide-based vaccines detailed above are also applicable to nucleic acid-based vaccines according to the invention, and all of the discussion above relating to routes and regimens for administration of polypeptides applies mutatis mutandis to nucleic acids. In addition, it is necessary to add that vaccines based on nucleic acids can be appropriately administered intravenously and intraarterially. Moreover, it is well known in the art that nucleic acid-based vaccines can be administered using a so-called gene gun, and therefore, this and equivalent methods of administration are considered part of the present invention. Finally, as reported, the use of nucleic acids MI M also gives good results, and therefore this particular method of administration is particularly preferred.

In addition, the nucleic acid(s) used as agents for immunization may contain coding regions of the 1st, 2nd and/or 3rd groups, for example, in the form of immunomodulatory substances described above, such as cytokines considered as suitable adjuvants. A preferred version of this implementation is associated with the coding region for the analogue and the coding region for the immunomodulator in different reading frames or at least under the control of different promoters. Thus, it is avoided that the analogue or epitope is produced as a fusion partner with the immunomodulator. Alternatively, two separate nucleotide fragments can be used, but this is less preferred due to the advantage provided by co-expression when both coding regions are included in one molecule.

Thus, the invention also relates to a composition for inducing the production of antibodies against APP or

AD, where the composition includes - a nucleic acid fragment or vector according to the invention (cf. discussion of vectors below) and - a pharmaceutically and immunologically acceptable carrier and/or adjuvant, as discussed above.

Under normal conditions, the nucleic acid encoding the variant is introduced in the form of a vector, where expression is under the control of a viral promoter. For a more detailed discussion of vectors according to the invention, see discussion below. Also, detailed descriptions related to the formation and use of vaccines based on nucleic acids | see Ooppeyu 9. ei a!., 1997, Appy. Kew. And so on. 15: 617-648 and BoppePu 99. ega!., 1997, Me bsiepsez 60: 163-172. Both reference data are incorporated herein by reference).

Live vaccines

The third alternative for the effective presentation of analogues to the immune system, as defined in options a-c, is the use of the method of live vaccines. In vaccination with live vaccines, presentation to the immune system is carried out by introducing non-pathogenic microorganisms into the animal, which can be transformed by a fragment of nucleic acid encoding an analog, or a vector that includes such a fragment of nucleic acid. The non-pathogenic microorganism can be any suitable attenuated bacterial strain (attenuated by passages or by removing pathogenic expressed products using recombinant DNA technology), for example, BSO. Musobasiegisht bromi5, non-pathogenic Zigeriosossi5 5yr., B. soii, ZaItopeia 5yr., Myrgio spoiegaye, ZPpideiia, etc. Reviews devoted to obtaining real live vaccines can be found, for example, in

Izaiioi R, 1995, Kemu. Damn 45: 1492-1496 and UmaIkeg RO, 1992, Massipe 10: 977-990, both of which are incorporated herein by reference).

For details on the nucleic acid fragments and vectors used in such live vaccines, see the discussion below.

As an alternative to bacterial live vaccines, a nucleic acid fragment of the invention discussed below can be introduced into a non-virulent viral vaccine vector, such as a cowpox strain or any other suitable smallpox virus.

Usually, a non-pathogenic microorganism or virus is injected into an animal only once, but in some cases it may be necessary to introduce the microorganism more than once during its life to maintain protective immunity. It is even suggested that the immunization schemes detailed above for polypeptide vaccination will be suitable for the use of live or viral vaccines.

Alternatively, vaccination with live vaccines or viruses is combined with previous or subsequent vaccination with polypeptides and/or nucleic acids. For example, primary immunization with a live or viral vaccine can be performed, followed by booster immunization using a polypeptide or nucleic acid-based approach.

A microorganism or virus can be transformed with nucleic acid(s) containing regions that encode groups 1, 2, and/or 3, for example, in the form of immunomodulatory substances described above, such as the cytokines discussed as suitable adjuvants. A preferred version of this implementation is associated with the coding region for the analogue and the coding region for the immunomodulator in different reading frames or at least under the control of different promoters. Thus, it is avoided that the analogue or epitope is produced as a fusion partner with the immunomodulator. Alternatively, two separate nucleotide fragments can be used as transforming agents. Of course, the presence of the 1st and/or 2nd and/or 3rd groups in the same reading frame can provide an analog according to the invention as an expression product, and such implementation is particularly preferred in the present invention.

Application of the method according to the invention for the treatment of a disease

As can be understood from the discussion above, providing a method according to the invention allows controlling diseases characterized by amyloid accumulation. In this context, AO is a key target of the method according to the invention, but other diseases characterized by amyloid deposits containing AD are also achievable targets. Therefore, an important implementation of the method according to the invention for the down-regulation of amyloid activity includes the treatment and/or prevention and/or improvement of the condition of AJ or other diseases characterized by the accumulation of amyloid, the method includes the down-regulation of APP or AD by the method according to the invention, to such an extent that the amount of amyloid is significantly reduced.

It is particularly preferred that the reduction of amyloid leads to a change in the balance between amyloid formation and degradation/removal, ie the rate of amyloid degradation/removal is increased to the extent that it exceeds the rate of amyloid formation. By carefully controlling the amount and immunological impact of the immunizations of the individual in need, a balance can subsequently be achieved that results in a net reduction in amyloid accumulation without excessive adverse effects.

Alternatively, if the method according to the invention is not effective in removing or reducing existing amyloid deposits in an individual, it can be used to produce a clinically significant reduction in the formation of new amyloid, thus significantly extending the period until the disease state does not deteriorate. It is necessary to be able to monitor the rate of amyloid accumulation either by measuring the serum concentration of amyloid (which is believed to be in equilibrium with the substance in the accumulations) or by means of a positron emission tomography (PET) scan, (cf. Ztai!

S.MU., ei ai., 1996, App. M.U. Asai 5si 802: 70-78.

Other diseases and conditions where these agents and methods can be used to treat or alleviate the condition in a similar way are mentioned above in the "prerequisites of the invention" or listed below in the section called "other amyloid diseases and proteins associated with them".

Peptides, polypeptides and compositions according to the invention

As can be seen from the above, this invention is based on the concept of immunization of individuals against an antigen

APP or AD to obtain a reduced number of pathology-related amyloid deposits. The preferred way to achieve such immunization is the use of analogs described here, thereby providing molecules not previously described in this area.

It is believed that the analogs discussed herein are the subject of the invention in themselves, and therefore an important part of the invention relates to the analogs described above. Therefore, any description presented herein relating to a modified APP or Ar is relevant for purposes of describing amyloidogenic analogs according to the invention, as well as any such descriptions are, conversely, applicable to the description of said analogs.

It should be noted that preferred modified APP or AD molecules include modifications that

which lead to the formation of a polypeptide having a degree of sequence identity with APP or AD of at least 7095, or with their subsequence at least 10 amino acids long. A higher degree of sequence identity, for example at least 75905 or even at least 80, 85, 90 or 9595 is preferred. The sequence identity for proteins and nucleic acids can be calculated as (Mg-Mg): 100/Mg, where Mg is the total number of non-identical residues in the two sequences when aligned and where Mg is the number of all residues in one of the sequences. Yes, the DNA sequence

ASTSASTS has a degree of identity with the sequence of AATSAATS, which is 7595 (Make2 and Me-8).

The invention also relates to compositions that can be used in the implementation of the method according to the invention. Therefore, the invention also relates to an immunogenic composition, which includes an immunogenically effective amount of the analogue described above, and said composition additionally includes a pharmaceutically and immunologically acceptable diluent and/or carrier, and/or filler and, optionally, an adjuvant . In other words, this part of the invention relates to compositions of analogues, mainly as described above. Thus, when the choice of adjuvants and carriers refers to the composition of modified or unmodified amyloidogenic polypeptide for use in the method according to the invention for the down-regulation of APP or AP, it is in accordance with what was discussed above.

Polypeptides are prepared according to methods well known in the art. Longer polypeptides are usually obtained using recombinant gene technology, which includes the introduction of an amino acid sequence encoding an analog into a suitable vector, transformation of a corresponding host cell with a given vector, expression of a nucleic acid sequence by a given host cell, extraction of the expression product from cells hosts or their supernatant and subsequent purification, and optionally additional modification, for example, refolding or derivatization.

Shorter peptides are preferably obtained using well-known methods of solid-phase or liquid-phase peptide synthesis. However, recent advances in this technology have made it possible to obtain full-length polypeptides and proteins using these methods, and therefore, obtaining long structures using synthetic methods is also not beyond the scope of this invention.

Nucleic acid fragments and vectors according to the invention

It is clear from the previous description that analogs of polyamino acids can be obtained with the help of recombinant gene technology, as well as with the help of chemical synthesis or semi-synthesis; The latter two alternatives are particularly relevant when the modification consists of binding to protein carriers (such as CI-H, diphtheria toxoid, tetanus toxoid and B5A) and non-bleaching molecules such as hydrocarbon polymers, and of course when the modification includes addition of side chains or side groups to the peptide chain obtained from APP or Ar.

For the purpose of recombinant gene technology, and of course for the purpose of nucleic acid immunization, nucleic acid fragments encoding analogs are important chemical products. Therefore, an important part of the invention relates to nucleic acid fragments encoding an analogue according to the invention, i.e. a polypeptide obtained from APP or AD, which includes either a natural sequence to which a fusion partner is added or inserted, or, preferably, a derived from APP or Ар polypeptide, in which a foreign T-cell epitope was introduced by means of insertion and/or addition, preferably by means of replacement and/or deletion. Nucleic acid fragments according to the invention are DNA or RNA fragments.

Nucleic acid fragments according to the invention are usually inserted into appropriate vectors to generate vectors that are cloned or expressed, carrying nucleic acid fragments according to the invention; such novel vectors also form part of the invention. Details relating to the construction of such vectors in accordance with the invention will be discussed below in connection with transformed cells and microorganisms. Vectors, depending on the purpose and type of application, can exist in the form of plasmids, phages, cosmids, minichromosomes or viruses, but "naked" DNA, which is transiently expressed only in certain cells, is also an important vector. Preferred cloning and expression vectors according to the invention are capable of autonomous replication, thus creating the possibility of obtaining a large number of copies for the purposes of high-level expression or high-level replication for subsequent cloning.

The basic design of a vector according to the invention includes the following elements in the 5-53 direction and in functional connection: a promoter for driving the expression of a nucleic acid fragment according to the invention, optionally a nucleic acid sequence that encodes a leader peptide that enables secretion (in the extracellular phase or, when applicable, in the periplasm) of the polypeptide fragment or its integration into the membrane, the nucleic acid fragment according to the invention and, optionally, the nucleic acid sequence encoding the terminator. When operating with expressing vectors in producer strains or cell lines for the purpose of genetic stability of the transformed cell, it is preferable that the vector, when introduced into the host cell, integrates into the genome of the host cell.

On the contrary, when working with vectors used for the expression of ip mimo in animals (for example, when using a vector in DNA vaccination), for safety reasons, it is preferable that the vector is incapable of integration into the genome of the host cell; usually use "naked" DNA or non-integrating vectors, the choice of which is well known to those skilled in the art.

Vectors according to the invention are used to transform host cells to obtain an analog according to the invention. Such transformed cells can be cultured cells or cell lines that are used for propagation of nucleic acid fragments and vectors according to the invention or used for recombinant production of analogs according to the invention. Alternatively, transformed cells may be live vaccine strains where a nucleic acid fragment (single or multiple copies) has been inserted to secrete or integrate the analogue into the bacterial membrane or cell wall.

Preferred transformed cells according to the invention are microorganisms such as bacteria (such as species of E. coli (e.g. E.

Musobrasiegiit Imostly non-pathogenic, for example, BSOo M. romi5|), yeasts (for example, sasspagotuse5 segemisiaee) and protozoa. Alternatively, the transformed cells are obtained from a multicellular organism, for example, a fungus, an insect cell, a plant cell, or a mammalian cell. Human derived cells are most preferred, see discussion of cell lines and vectors below. The latest results in the authors' laboratory indicate the prospects of using the commercially available line Ogozorpya teiIapodavieg (cell line and vector system Zsppeideg 2 (552), available in Ipmigodep) for recombinant production of polypeptides, and therefore, this expressing system is particularly preferred.

For purposes of cloning and/or optimized expression, it is preferred that the transformed cells are capable of replicating the nucleic acid fragment according to the invention. Cells expressing a nucleic acid fragment are particularly useful embodiments of the present invention; they can be used for the limited or large-scale preparation of an analog according to the invention or, in the case of non-pathogenic bacteria, as vaccine components in a live vaccine.

When obtaining analogs according to the invention with the help of transformed cells, it is convenient, although it is far from the essence, that the expression products are either exported to the outside in the culture medium, or are located on the surface of the transformed cell.

When effective producer cells are identified, it is preferable to create a stable cell line based on them that carries a vector according to the invention and expresses a nucleic acid fragment encoding a modified amyloidogenic polypeptide. Preferably, a given stable cell line secretes or carries an analog according to the invention, thus facilitating its purification.

As a rule, plasmid vectors containing replicon and control sequences obtained from species compatible with the host cell are used in connection with the hosts. The vector usually carries a replication site as well as marker sequences capable of selecting transformed cells by phenotype.

For example, E. soya is commonly transformed using rvVK322, a plasmid obtained from the species E. soya

For example, see Voyimag eyi a!., 1977). Plasmid rvk.322 contains ampicillin and tetracycline resistance genes and thus provides a simple means of identifying transformed cells. The rve plasmid or other microbial or phage plasmid must also contain - or be modified to contain - promoters that the prokaryotic microorganism can use for expression.

Such promoters, which are most often used for the construction of recombinant DNA, include the B-lactamase and lactose promoter systems (Spapo et al., 1978; MKakiga et al., 1977; Soedavi! et al., 1979| and the system of tryptophan (games) of the promoter |Soedaeii! ei ai., 1979; ER-A-0036116)|. Although they are the most commonly used, other microorganism promoters have also been discovered and used, and details of their nucleotide sequences have been published, allowing specialists to functionally ligate them with plasmid vectors (IbZierzhepiiv ei ai!., 19801. The identified prokaryotic genes can be efficiently expressed in E. soybean from its own promoter sequence, preventing the need to add another promoter by artificial means.

In addition to prokaryotic, eukaryotic microorganisms such as yeast cultures can also be used, and here the promoter must be able to direct expression. Zasspagotuse5 sehemiziasis, or common baker's yeast, from eukaryotic microorganisms is used most often, although a number of other strains are also available. For expression in Zasspagotus5, for example, the UKr7 plasmid is usually used

IZiippsot ei a/!., 1979; Kipdztap eyi a!., 1979; Tespetreg" ei a!., 1980). This plasmid already contains the igri gene, which is a selective marker for a mutant strain of yeast deprived of the ability to grow on tryptophan, for example, ATS Mo44076 or PERA-1 | Chopez, 1977). The presence of damage to the igri as characteristics of the host yeast cell genome, provides an effective condition for detection of transformation by growth in the absence of tryptophan.

Suitable promoter sequences in yeast vectors include promoters for 3-phosphoglycerate kinase (Nez et al., 1980) or other glycolytic enzymes (Nez et al., 1968; NoPapa et al., 1978))| such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphatisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase and glucokinase. In order to ensure polyadenylation of mRNA and termination, it is desirable that when constructing appropriate expressing plasmids, terminating sequences associated with these genes are also ligated into the 3'-end of the expressing vector sequence.

Other promoters that have the additional advantage of transcription controlled by growth conditions are the promoter regions of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradation enzymes associated with nitrogen metabolism and glyceraldehyde-3-phosphate dehydrogenase indicated above, and enzymes responsible for maltose utilization and galactose. Any plasmid vector containing a promoter suitable for yeast, an origin of replication and a terminator sequence is suitable.

In addition to microorganisms, cell cultures obtained from multicellular organisms can be used as hosts. In principle, any such cell culture is possible, whether it is a culture from vertebrates or invertebrates. However, cells of vertebrates are of greatest interest, and propagation of vertebrate cells in culture (tissue culture) has become a common method in recent years (Tizvye Sishge, 1973).

Examples of such suitable host cell lines are MEKO and Ne! a, Chinese Hamster Ovary (CHO) and UM138, BNK, CO5-7 293 cell lines, Zrodoriega iadiregda (ZE) cells (commercially available as complete systems expressing, among other things, in Rgoieip Zsiepse5, 1000 Kezeagsp Ragkulau, Megidep , ST 06450, ).5.A. and in Ipmigodep) and MOSCOW cell lines. The most preferred cell line according to this invention is line 52, available at Ipmigodep, RO Vokh 2312, 9704 SN Sgopipdep, Tpe Meipepapav5.

Expression vectors for such cells typically include (if necessary) an origin of replication, a promoter located upstream of the gene to be expressed, together with any necessary ribosome binding sites, RNA splicing sites, a polyadenylation site, and transcription terminator sequences. .

For use in mammalian cells, control functions on expression vectors are often provided by viral material. For example, commonly used promoters are obtained from the polyoma virus, adenovirus 2 and, most often, simian virus 40 (5M40). The early and late promoters of the 5M40 virus are particularly useful due to the fact that they are easily obtained from the virus as a fragment that also contains the site of the start of replication of the 5M40 virus (Rieg5 ei a!., 1978). The smaller and larger 5M40 fragments can also be used, provided that a sequence of approximately 250 bp is included that extends from the Nipai recognition site to the VaiY recognition site located in the viral origin of replication. In addition, it is also possible and often desirable to use a promoter or control sequences, usually associated with the desired gene sequence, provided that these control sequences are compatible with host cell systems.

The origin of replication can be provided either by engineering the vector to include an exogenous origin of replication, for example, which can be obtained from 5M40 or other viruses (eg, polyomavirus, adenovirus, M5M, BPM), or by a mechanism of chromosomal host cell replication. If the vector is integrated into the chromosome of the host cell, it is often sufficient.

Identification of suitable analogues

It is clear to those skilled in the art that not all possible variants or modifications of natural APP or AR have the ability to cause the formation of antibodies in an animal that are cross-reactive with the natural form.

However, it is not difficult to perform an efficient standard screening of modified amyloidogenic molecules that satisfy the minimum requirements of immunological reactivity discussed here. Therefore, it is possible to apply a method for the identification of modified amyloidogenic polypeptides capable of inducing the formation of antibodies against an unmodified amyloidogenic polypeptide in animal species, where the unmodified amyloidogenic polypeptide is a (non-immunogenic) own protein, and this method includes - obtaining by means of peptide synthesis or methods of gene engineering a set of mutually simple analogs according to the invention, wherein amino acids are added, inserted, deleted, or substituted in the amino acid sequence of the APP or AD of the animal species, thereby resulting in amino acid sequences in the set that include T-cell epitopes , foreign to the animal species, or obtaining a set of nucleic acid fragments encoding a set of mutually simple analogs, - elements of testing a set of analogs or nucleic acid fragments for their ability to cause the production of antibodies in an animal species against unmodified APP or Ar, - and identification and, neo necessarily, the selection of element(s) of the set of analogs that significantly induces in the species the production of antibodies against unmodified APP and AD or the identification or, optionally, the selection of products expressing the polypeptide encoded by the elements of the set of nucleic acid fragments, that significantly induce the production of antibodies against unmodified APP and AD in the animal species.

In this context, a "set of mutually simple modified amyloidogenic polypeptides" is a collection of non-identical analogs selected based on the criteria discussed above (eg, in combination with circular dichroism studies, NMR spectra, and/or X-ray diffraction profiles). A set can consist of only a small number of elements, but it is believed that a set can contain several hundreds of elements.

The testing of kit elements can ultimately be done by IP mimo, but a number of IP migo tests can be used that reduce the number of modified molecules that serve the purpose of this invention.

Since the purpose of introducing foreign T-cell epitopes is to support a B-cell response by T cells, a prerequisite is that the analogue induces T-cell proliferation.

Proliferation of T cells can be tested using standardized ip migo proliferation assays.

Briefly, a T-cell-enriched sample is obtained from the subject and subsequently maintained in culture. Cultured T-cells are brought into contact with the subject's APC, which has previously taken up the modified molecule and converted it for presentation to T-cell epitopes. The proliferation of T cells is observed and compared to an appropriate control (eg, T cells in culture contacted with APC that converted intact native amyloidogenic polypeptide). Alternatively, proliferation can be measured by measuring the concentration of release of appropriate cytokines by T cells in response to their recognition of foreign T cells.

It seems highly probable that since at least one analog of each type of set is capable of inducing the production of antibodies against APP or Ar, it is possible to obtain an immunogenic composition that includes at least one analog capable of inducing the formation of antibodies against unmodified APP or Ar in an animal species where unmodified APP or AD are their own proteins, in a way that involves mixing element(s) of the set, which significantly induces the production of antibodies in an animal species that reacts with

APP or AR with pharmaceutically and immunologically acceptable carriers and/or solvents and/or diluents and/or fillers, optionally in combination with at least one pharmaceutically and immunologically acceptable adjuvant.

The above-discussed tests of sets of polypeptides are readily performed by initially obtaining a number of mutually simple nucleic acid sequences or vectors according to the invention, inserting them into appropriate expression vectors, transforming appropriate host cells (or animal hosts) with the vectors, and expressing the nucleic acid sequences according to the invention. These stages can be followed by the release of expression products. Preferably, nucleic acid sequences and/or vectors are obtained by methods that include the use of molecular amplification methods, such as PCR or by nucleic acid synthesis.

Specific amyloidogenic targets

In addition to the most commonly associated with Alzheimer's disease proteins APP, ApoEA4 and Tac, there is a long list of other proteins, one way or another associated with AI either due to their direct presence in plaques and tangles in the brain in AD or due to their pronounced genetic association with increased risk development of JSC. Most, if not all, of these antigens, along with AB, APP, presenilin and ApoE4 discussed above, are possible target proteins in certain embodiments of this invention. These possible targets have already been comprehensively discussed in (MO 01/62284|. Therefore, these possible targets will only be briefly mentioned here, while a more detailed and thorough discussion can be found in IMO 01/62282|, incorporated herein by reference: alpha!1- antichymotrypsin (AST); alpha2-macroglobulin; AVAO (alcohol dehydrogenase, which binds Ar-peptide); ARI RI and -2 (protein similar to amyloid precursor protein 1 and 2); AMU117; Vach; Vsy-2; hydrolase bleomycin; BEI/AVEII; chromogranin A; clusterin/arrow; CEE-binding protein (corticotropin-releasing factor); EOTE (endothelium-derived toxic factor); heparan sulfate proteoglycans; human collapsin response mediator protein 2; gentingin ( Huntington's disease protein); ISAM-1Y; I--6; CO68 antigen associated with lysosomes; P21 gas; Ri C-delta 1 (isoenzyme delta 1 of phospholipase C); component of serum amyloid P (ZAR); synaptophysin; synuclein (alpha -synuclein or MACP) and TOBE-B1 (transforming growth factor p1).

The means and methods of down-regulation of APP or AD described herein can be combined with treatment, for example, active specific immunotherapy against any of these other amyloidogenic polypeptides.

In addition to Alzheimer's disease, cerebral amyloid angiopathy is also a disease that may be a suitable target for the method provided herein.

It is suggested that most methods of immunization against APP or Ar should be limited to immunizations that lead to the formation of antibodies that cross-react with native APP or Ar. However, in some cases, it is of interest to induce cellular immunity in the form of STIs - responses against cells that present MHC class I epitopes from amyloidogenic polypeptides - this may be appropriate in cases where the reduction in the number of cells producing APP or Ar does not bring serious adverse effect In those cases when STI is the answer, it is preferable to use the recommendations of the authors of the publication (MLO 00/20027). The descriptions of said two documents are therefore incorporated herein by reference.

Immunogenic carriers

Molecules that include a T-helper epitope and APP or AD peptides that are or include B-cell epitopes covalently linked to a non-immunogenic polymer molecule that acts as a carrier, such as a polyvalent activated polyhydroxy polymer, can be prepared that will , as indicated above, function as vaccine molecules containing only the immunologically relevant parts, and they represent the implementation of interest in the variants 4 and are discussed above. It is possible to use mixed or so-called universal T-helper epitopes, for example, if the target for the vaccine is its own APP or AD protein. In addition, the elements that enhance the immunological response can also be linked together with the carrier and thus they will act as an adjuvant. Such elements can be mannose, tuftsin, muramyl dipeptide, Spo motifs, etc. In this case, further adjuvant formulation of the vaccine product may not be necessary, and the product may be administered in pure water or saline.

By binding epitopes of cytotoxic T cells (CTIs) to T-helper epitopes, it is possible to generate

STIs, specific in relation to the antigen from which the STI is derived -epitope. Elements that facilitate the capture of the product in the cytosol of APC, for example, macrophages, such as mannose, can also bind to the carrier, along with STI - and T-helper epitopes, and enhance the STI response.

The ratio of B-cell and T-helper epitopes (P2 and RZO) in the final product can be varied by changing the concentration of these peptides at the synthesis stage. As indicated above, the immunogenic molecule can be labeled with, for example, mannose, tuftsin, Cro-motifs or other immunostimulatory substances (described herein) by adding them, if necessary, with the help of, for example, aminated derivatives of these substances, to the carbonate buffer at the synthesis stage.

If an insoluble activated polyhydroxypolymer is used to combine peptides that contain B-cell and T-helper epitopes of APP or AD, then this can, as indicated above, be carried out in the form of solid-phase synthesis, and the final products can be collected and purified by washing and filtration.

Elements for binding to the activated tresyl polyhydroxypolymer (peptides, labels, etc.) can be added to the polyhydroxypolymer at low pH, for example, at pH 4-5, and allowing them to redistribute evenly in the "gel" by passive diffusion. Next, the pH can be raised to pH 9-10 to trigger the reaction of the basic amino groups on the peptides and the tags with tertyl groups on the polyhydroxypolymer. After the binding of peptides and, for example, immunostimulating elements, the gel is crushed to form particles of the size suitable for immunization. Such an immunogen thus includes: a) at least one first amino acid sequence derived from APP or AD, where at least one first amino acid sequence contains at least one B-cell and/or at least one STi epitope, and

B) at least one second amino acid sequence, which includes a foreign epitope of T-helper cells, where each of at least the first and at least the second amino acid sequences are linked to a pharmaceutically acceptable activated hydroxypolymer carrier.

In order to bind amino acid sequences to a polyhydroxypolymer, it is usually necessary to "activate" the polyhydroxypolymer with a suitable reactive group that can form the necessary bond with the amino acid sequences.

It is understood that the term "polyhydroxypolymer" has the same meaning as in (MO 00/053161|, i.e. the polyhydroxypolymer can have exactly the same characteristics as specifically indicated in this application. Therefore, the polyhydroxypolymer can be water-soluble or water-insoluble (such thus requiring different stages of synthesis in the process of receiving the immunogen.) The polyhydroxy polymer can be selected from natural polyhydroxy compounds and synthetic polyhydroxy compounds.

Specific and preferred polyhydroxypolymers are polysaccharides selected from acetate, amylopectin, agar-agar gum, agarose, alginates, gum arabic, carrageenan, cellulose, cyclodextrins, dextran, furcellaran, galactomannan, gelatin, dpaya, glucan, glycogen, guar, Cagua, copias /A, resins of carob fruits, mannan, pectin, rzuiiisht, riishiap, starch, iatagipe, tragacanth, xanthan, xylan and xyloglucan. Dextran is particularly preferred.

However, the polyhydroxy polymer can also be selected from highly branched poly(ethyleneimine) (REI), tetrathienylenevinylene, Kevlar (long chains of polyparaphenylterephthalamide), poly(urethanes), poly(siloxanes), polydimethyl siloxane, silicone, poly(methyl methacrylate) (PMMA), polyvinyl alcohol ), poly(vinylpyrrolidone), poly(2-hydroxyethyl methacrylate), poly(M-vinylpyrrolidone), polyvinyl alcohol), poly(acrylic acid), polytetrafluoroethylene (PTEE), polyacrylamide, poly(ethylene covinyl acetate), poly(ethylene glycol) and derivatives, poly(methacrylic acid), polylactides (RIGA), polyglycolides (RA), poly(lactide coglycolides) (RI. CA), polyanhydrides and complex polyorthoesters.

The average molecular weight of the polyhydroxypolymer under consideration (for example, before activation) is usually at least 1000, for example at least 2000, preferably in the range of 2500-2000000, more preferably in the range of 3000-1000000, especially 5000-500000. The examples show that polyhydroxy polymers with an average weight in the range of 10,000-200,000 are particularly advantageous.

The polyhydroxy polymer is preferably soluble in water at a concentration of at least 1 Ω/ml, preferably at least 25 mg/ml, for example at least 500 mg/ml, especially at least 10 Ω/ml, for example at least 150 mg/ml at room temperature. Dextran, even when activated as described herein, is known to meet water solubility requirements.

For some of the most interesting polyhydroxy polymers, the ratio between C groups (carbon atoms) and

OH (hydroxyl groups) of unactivated polyhydroxy polymers (i.e., native polyhydroxy polymers before activation) is in the range from 1.3 to 2.5, for example, 1.5-2.3, preferably - 1.6-2.1, especially - 1, 85- 2.05. Without being bound by any particular theory, it is believed that this C/OH ratio of the unactivated polyhydroxypolymer represents the most favorable level of hydrophilicity. Polyvinyl alcohol and polysaccharides are examples of polyhydroxy polymers that meet this requirement.

It is believed that the above ratio should remain approximately the same for the activated polyhydroxy polymer, while the activation ratio should be significantly lower.

The term "polyhydroxypolymer carrier" is intended to refer to the region of the immunogen that carries the amino acid sequence. As a general rule, the polyhydroxypolymer carrier has its outer boundaries where amino acid sequences can be cleaved by peptidases, for example, in an antigen-presenting cell that processes an immunogen. Therefore, the polyhydroxypolymer carrier can be a polyhydroxypolymer with an activation group, where the bond between the activation group and the amino acid sequence is cleaved by peptidases in APC, or the hydroxypolymer carrier can be a hydroxypolymer with an activation group and, for example, a bridge, such as a single I -amino acid or a series O- amino acids, where the last part of the bridge can bind amino acid sequences and be cleaved by peptidases in APC.

As indicated above, polyhydroxy polymers carry functional groups (activating groups) that facilitate the fixation of peptides on the carrier. In this area, a huge number of applicable functional groups are known, for example, tresyl (trifluoroethylsulfonyl), maleimide, para-nitrophenylchloroform, cyanobromine, tosyl (para-toluenesulfonyl), trifyl (trifluoromethanesulfonyl), pentafluorobenzenesulfonyl, and vinylsulfonyl groups. Preferred examples of functional groups according to the present invention are tresyl, maleimide, tosyl, trifyl, pentafluorobenzenesulfonyl, para-nitrophenylchloroform and vinylsulfone groups, among which tresyl, maleimide and tosyl groups are particularly significant.

Activated tresyl polyhydroxy polymers can be obtained using tresyl chloride, as described for the activation of dextran in example 1 and MO 00/05316 or as described in sedogichchch5 ei ai., 9. Ittipoi.

May 181 (1995) 65-73).

Maleimide-activated polyhydroxy polymers can be prepared using /para-maleimidophenylisocyanate as described for the activation of dextran in (Example C in M/O 00/05316).

Alternatively, maleimide groups can be introduced into a polyhydroxy polymer, such as dextran, by derivatization of a tresyl-activated polyhydroxy polymer (e.g., tresyl-activated dextran (TAO)) with an excess of a diamine compound (typically NaM-CaoNop-MnO, where n is 1-20, preferably 1-8), for example, 1,3-diaminopropane, and, subsequently, by the reaction of the amino groups introduced into TAO with reagents such as succinimidyl-4-(M-maleimidomethyl)cyclohexane-1-carboxylic acid (5MCOS), sulfosuccinimidyl-4-( M-maleimidomethyl)cyclohexane-1-carboxylic acid (sulfo5MCS), succinimidyl-4-(para-maleimidophenyl)butyrate (5MRV), sulfosuccinimidyl-4-(para-maleimido-phenyl)butyrate (sulfob5MRV), MI-y- maleimidobutyryloxysuccinimide esters (CMV5) or M-y-maleimidobutyryloxysulfosuccinimide esters. Although different reagents and activation pathways formally lead to slightly different maleimide-activated products with respect to the relationship between the maleimide functionality and the remaining parent hydroxyl group on which the activation was performed, they can all be collectively and individually considered "maleimide-activated polyhydroxy polymers".

Activated tosyl polyhydroxy polymers can be obtained using tosyl chloride as described for the activation of dextran in (Example 2 in UMO 00/05316). Polyhydroxypolymers activated with trifyl and pentafluorobenzenesulfonyl are prepared as analogs activated with tosyl or tresyl, for example, using the appropriate acid chlorides.

Cyanogen bromide-activated polyhydroxypolymers can be obtained by reacting polyhydroxypolymer with cyanogen bromide using traditional methods. The resulting functional groups are usually esters of cyanic acid with two hydroxyl groups of the polyhydroxypolymer.

The degree of activation can be expressed as the ratio between free hydroxyl groups and activation groups (that is, hydroxyl groups that have become functional). It is believed that the ratio between the free hydroxyl groups of the polyhydroxypolymer and the activation groups to obtain a favorable balance between the hydrophilicity and reactivity of the polyhydroxypolymer should be from 250:1 to 4:1. Preferably, the ratio is from 100:1 to 61, more preferably from 60:1 to 871, especially from 40:1 to 10:1.

Particularly interesting activated polyhydroxy polymers for use in the method for obtaining an immunogen applicable in most cases according to the invention are activated trezilom,

tosyl and maleimido polysaccharides, especially activated tresyl dextran (TA), activated tosyl dextran (TO5AO) and activated maleimido dextran (MAB).

Preferably, the bond between the polyhydroxypolymer carrier and the amino acid sequences associated with it is cleaved by peptidases, for example, such as peptidases active in the processing of antigens in ARS. Therefore, it is preferable that at least the first and at least the second amino acid sequences are linked to the activated polyhydroxypolymer carrier by means of an amide or peptide bond. It is particularly preferred that each of at least the first and at least the second amino acid sequence provides a nitrogen group for the amide bond corresponding to it.

The polyhydroxypolymer carrier may be free of amino acid residues if the activation group is required to provide a peptidase-cleavable linkage portion, but as indicated above, the carrier may simply include a spacer including at least one I-amino acid. However, at least the first and at least the second amino acid sequence is usually linked to the activated version of the polyhydroxypolymer by means of nitrogen at the M-terminus of the amino acid sequence.

The immunogen described above, which is used in most cases, according to this invention can be used in methods of immunization, in fact, as described herein, for polypeptide vaccines. That is, all descriptions discussed here relating to doses, methods of administration and composition of polypeptide vaccines for down-regulation of amyloidogenic polypeptides are used with appropriate modifications for commonly used immunogens.

A safe method of vaccination is usually used

As discussed above, one of the preferred embodiments of the present invention involves the use of variants of amyloidogenic peptides unable to present their own Tn epitopes capable of directing an immune response against an amyloidogenic polypeptide.

However, the authors of this invention believe that this strategy for the development of autovaccines and for the induction of autoimmunity represents a technology applicable in most cases, which is an object of the invention in itself. It should be considered particularly convenient cases when the autoantigen sought for downregulation is present in the body in a significant excess, so that it is possible that autostimulation of the immune response may occur. Therefore, all of the above descriptions of this embodiment, as it relates to the provision of an autoimmune response against APP or AB, are, with appropriate modifications, applicable to immunization against self-polypeptides, especially those that are present in substantial amounts to maintain an immune response in the form of an uncontrolled autoimmune condition due to the fact that Tn-epitopes of the corresponding own proteins direct the immune response.

Example 1

Autovaccination approach for immunization against AYU

The fact that AD protein gene knockout mice show no abnormalities or adverse side effects indicates that eliminating or reducing AD levels is safe (Apepo N. (1996)).

Published experimental data, where transgenic animals were immunized against the transgenic human Aβ protein, indicate that if it is possible to break autotolerance, then the downregulation of Aβ can be obtained with the help of autoreactive antibodies. These experiments, in addition, indicate that such down-regulation of AD could potentially both prevent the formation of plaques and clear the brain of already formed Ar-plaques, |cf. ZspepkK ei ai. (19991). However, it is usually impossible to obtain antibodies against one's own proteins.

Thus, published data do not provide ways to break true self-tolerance to true self-proteins. The data also do not provide information on how to ensure, if necessary, that the immune response is directed exclusively or preferentially against Aβ deposits, and not against the cell membrane-bound AD precursor protein (APP). The immune response obtained with the existing technology will preferentially cause an immune response against self-proteins in an unregulated manner, so that unwanted or excessive autoreactivity against areas of the AD protein can be obtained. Therefore, with the use of existing immunization strategies, it is most likely impossible to obtain strong immune responses against one's own proteins; moreover, it is dangerous due to potential strong cross-reactivity with the cell membrane-bound

APP, which is present in a large number of CNO cells.

The present invention provides methods of effectively generating a strong immune response against true self-proteins that can potentially form plaques and cause serious disease of the CNS or other parts of the body. With the use of this technology, a safe and effective therapeutic vaccine based on AD protein will be developed for the treatment of AJ.

In light of this, it can be expected that AbO, a disease predicted to wreak havoc on the health care system in the next century, can be cured; or such described vaccines may at least provide an effective therapeutic approach to treating the symptoms and progression of a given disease.

This method is a completely new immunological approach to blocking the accumulation of amyloid in

AB, as well as in other neurological diseases.

The table below lists the 35 designs under consideration. All positions are given in the table relative to the starting methionine of APP (the first amino acid in 5ZEO IU MO:2) and include both starting and ending amino acids, for example, the fragment 672-714 includes both amino acid 672 and 714. Starting and ending positions for P2 and RZO show that the epitope replaces a part of the APP fragment in the indicated positions (both positions are included in the replacement), in most of the constructions the introduced epitopes replace a fragment equal in length to the epitope. Asterisks in the table mean the following: 5) Only one position for Ра and RZO indicates that in this position the epitope was inserted into the derivative

APP (the epitope begins with an amino acid adjacent to the specified position from the C-end). 5) Construction 34 contains three identical APP fragments separated by RZO and Pg, respectively. construct 35 contains nine identical APP fragments separated by RZO epitopes and

P2 alternating.

Constructions of APR Asho Mas

The beginning of the fragment | End of fragment I Position of the epitope

APP APP APP

3 | .юЮюКМ,вб/2 | 77ryuryuIyuIyuyu | 735749, | 714728. | 99 4 | .Yuyurup6vl | |1111111717171717171С1С1С1С1 4728799 | щЦщРб/2 | 77/71 ro 7-78 |77777171717171111111111111р99 6 | .shyuYushКХ bla | 7777/7770 | 7777/7231 |11717171sta3 | 135 7 | 77717672 | 77770 | 77777777 th 8 | юЮюющ 6/2 | .-.YuyuYuYuYy |ta |1117|1f ma 9 | юЮюющ 6/2 | -HB 4 |. 777777777С7С7С7С7С7С7С7С7|77717DDs6uye moat | 77777672 | 777774. vl |. |about 14 | 62 | 74 | 680-694 | -::K ...//O| 43 14 | 672 | 714 | 685-799,.474Й | 2 .YUY.K.:./km6ny/ | 43 716 | Yuk, 6/2 | 7/4 | 690704 | .....Скк | 4 17 | ...yuryu6/2 | ry | 695-709. | 7 az 18 | ..YuyuYuyu«Й6bl2 | 7777/7477 675-695. /2 ... | ..Dudyudyusplla 17777777717171717171717171717171717171717171717171717171717171717171717111111111116OO -YO | With 26 | - BU | O 774 | 777777777777 2 | 77/74 | 17717111717171717111111111111t7oi1111 trench 29 | B 6/2 | 74 |; 77777771 trench | .-.KhGU(bl2 | 77/74 | 7777 68717711 other pieces of SE PO S ETNO OPO2 PO - s ZNN - 6 3 /2 | 77/41 11111111111111111rovv 33 | bl | 7/4 77711111 11111111

The area of APP against which it is most interesting to get an answer is 43 amino acids of the AD cortical peptide (AD-43, which corresponds to ZEO IU MO:2, residues 672-714), which is the main component of amyloid plaques in the brain in AU. This fragment of APP is a part of all the structures listed above.

Variants 1 and 2 contain the area of APP upstream of transcription from Ar-43, where model epitopes of Ra and

RZO All options 1 and 3-8 contain a C-100 fragment, which is shown to be neurotoxic - the C-100 fragment corresponds to amino acid residues 714-770 of 5ZEO IJOO MO:2. In variants 3-5, the epitopes replace part of the C-100 fragment, while in variants 6-8, the epitopes were inserted into C-100.

Variants 9-35 contain only bovine protein Ar-43. In options 9-13, P2 and RZO are merged with one or the other end

Ar-43; in 14-21 P2 and RZO replace part of Ar-43; in 22-33 P2 and RZO were inserted into Ar-43; 34 contains three identical fragments of Ar-43, separated by RZO and Rg, respectively; 35 contains 9 repeats of Ар-43, separated by epitopes Ра and РЗО, alternating.

In accordance with this invention, immunogenic analogues can also use shortened parts of the AD-43 protein discussed above. Shortened parts of AD(1-42), AD(1-40), AD(1-39), Ар(1-35), АД(1-34), Ар(1-28), Ар(1 -12), Ar(1-5), AD(13-28), Ar(13-35), Ar(17-28), Ar(25-35), Ar(35-40),

Ar(36-42), and Ar(35-42) (where the numbers in parentheses indicate the amino acid sections of Ar-43 that make up the corresponding fragment, for example, Ar(35-40) is identical to amino acids 706-711 in 5EO IU MO:2 ). All of these variants with shortened parts of ArD-43 can be obtained with AD fragments described here, especially with variants 9, 10, 11, 12, and 13.

In some cases, it is preferable that Ar-43 or its fragments are mutant. Substitution variants in which methionine at position 35 of Ar-43 is replaced mainly by leucine or isoleucine or simply deleted are particularly preferred. Particularly preferred analogs contain a single methionine located at the C-terminus, or because it is naturally occurring in an amyloidogenic polypeptide or foreign epitope

Tn, or due to the fact that it was inserted or added. Therefore, it is also preferred that the portion of the analog that includes the foreign Tn epitope be free of methionine, except for the possible C-terminal location of methionine.

In fact, it is generally preferred that all APP or Aβ analogs used in accordance with the present invention have the common characteristic of simply including a single methionine located in the analog as the C-terminal amino acid, and all other methionines as in the amyloidogenic polypeptide, so and in the foreign Tn epitope, were deleted or replaced by another amino acid.

An additional mutation of interest is the deletion or substitution of phenylalanine at position 19 in ARD-43, and it is particularly preferred that this mutation represents the replacement of this phenylalanine residue with proline.

The following table defines a group of particularly preferred constructions that function with shortened forms or mutations of Ar-43: mene ve He | WHERE IS THE BAG?

Me variant| used in the molecule, AR relative to 1 ac P2 relative to 1 ac RZO relative to 1 ac length relative to 1 ac AD (1-42/43) molecules molecules molecules molecules (ac)

In this table, the AD segment used in the molecule is indicated by amino acid numbers relative to 1 ac of the AD molecule (1-42/43), i.e. 1-28 means that a fragment was used in the molecule

AB (1-42/43) 1-28. If two or more different fragments were used, both are indicated in the table, i.e. 1-12 (a)-13-28 (p) means that the molecule used both the fragment Ar (1-42/43) 1-12 and the fragment 13-28.

Also, if the same segment is present in the construct in more than one copy, this is indicated in the table, i.e. 1-12 (x3) indicates that the AD fragment (1-42/43) 1-12 is present in the construct in three copies

Next, the position of the AD segment in the molecule is indicated by the amino acid positions relative to the first amino acid in the molecule, i.e. 22-49 indicates that the AD fragment in question is located in the molecule from amino acid 22 to amino acid 49 inclusive. Position of epitopes P2 and

RZO are marked in the same way. If two or more different AD fragments were used in the molecule, all their positions are indicated, i.e. 1-12 (a)-49-64 (p) means that fragment (a) is located in the molecule from ac 1 to ac 12, and fragment ( B) - from AK 49 to 64.

Moreover, if the molecule contains more than one copy of the same fragment, the indicated positions of all copies, i.e. 1-12, 34-45, 61-72, shows that three copies of the AD fragment are located in the molecule in positions 1-12, 34 -45 and 61-72, respectively.

Finally, the indication of the total length of each molecule includes both AD fragment(s), and P2 and RZO epitopes.

Variant 42 contains two amino acid substitutions at positions 19 (rpe to rgo) and 35 (thei to uz), as indicated in the column representing Ar fragments.

See Figure 1 and the table above for details of specific points of introduction of foreign T-cell epitopes.

One of the additional construction types is particularly preferred. Since one of the objectives of the present invention is to avoid destruction of APP-producing cells, while elimination of Aβ is desirable, it is considered suitable to obtain autovaccine designs that include only those portions of Aβ that, when present in

APP, are not exposed in the extracellular phase. Thus, similar designs must contain at least one B-cell epitope derived from the amino acid fragment defined by amino acids 700-714 in 5EO IU MO:2. Since it is predicted that such a short polypeptide fragment will be only weakly immunogenic, it is preferable that such an autovaccine construct consists of several copies of the B-cell epitope, for example, in the form of a construct having the structure shown in formula 1 in the detailed description of this invention, cf. above. In this version of formula 1, the terms amyloidei-amyloidech represent the x B-cell epitope containing the sequence of amino acids obtained from amino acids 700-714 5EO IU MO:2.

The preferred alternative is the possibility described in detail above of linking the amyloidogenic (polysupeptide) with the selected T-helper epitope by means of an amide bond with the polysaccharide carrier molecule, in this way multiple presentations of the "weak" epitope constructed by amino acids 700-714 of 5EO IU become possible MO:2, and it also becomes possible to choose the optimal ratio between B-cell and T-cell epitopes.

Example 2

Immunization of transgenic mice with Ar and modified proteins according to the invention.

Construction of DNA encoding PAV43--34. The pPAV43--34 gene was constructed in several stages. First, a PCR fragment was obtained with primers MEMe801 (5EBEO 0 MO:10) and MEM»802 (5EO 10 MO-:11), using the primer MEMe800 (ZEO 10 MO:9) as a matrix. MEMe800 encodes a fragment of human area-43 with codons optimized for E. soya. MEMe801 and 802 add corresponding restriction sites to the fragment.

The PCR fragment was purified and cleaved by Msoyi! and Nipaii, again purified and cloned in split Meoi1-

Nipa and the purified vector for expression in E. soybean PET28r. The resulting plasmid encoding wild-type human ArD-43 was named pAV'1.

At the next stage, the T-helper epitope P2 was added to the C-end of the molecule. Primer MEMe806 (5EO IU

MO:12) contains the sequence encoding the P2 epitope, thus, the fusion product of Pα and areba-43 is obtained using the PCR reaction.

Cloning was performed by obtaining a PCR fragment with primers MEMe178 (5EO IU

MO:8) and MEMo806, using rAVI1 as a matrix. The fragment was purified, cleaved with Misoi and Nipa, purified again and cloned into the cleaved Meo1I-Nipa!y and the purified pET28r vector. The resulting plasmid was called pAV2.

In a similar way, another plasmid was obtained, which carries the coding sequence of Ar-43 with another T-

helper epitope, RZO, added to the M-end. This was done by obtaining a PCR fragment with primers MEMe105 (ZEO IU MO:7) and MEMo807 (5EO IO MO:13), using rAVI1 as a template.

The fragment was purified, cleaved in Mesoi and Nipaii, purified again and cloned in cleaved Meoi1-

Nipa and the purified vector pET28r. The resulting plasmid was named pAV3Z.

At the third stage, the second repeat of Ar-43 from the C-end was added to the epitope P2 of plasmid pAV2 using the primer MEMo809 (5EO 10 MO:14). At the same time, MEMo809 creates a Watni site immediately after repeating Ar-43. The PCR fragment was obtained with primers MEMe178 and MEMe809, using pАВ2 as a matrix. The fragment was cleaved with Meso1 and NipaI, purified and cloned into the cleaved Meso1-NipaI and the purified pET28r vector. This plasmid was called rAVaA.

Finally, the sequence of the RZO epitope - the repeat of Ар-43 from pАВЗ was cloned into the plasmid pАВ4. This was done by obtaining a PCR fragment with primers MEMe811 (ZEO IU MO:16) and MEMe105, using it as a matrix of rAVZ. The fragment was purified and used as a primer in subsequent PCR with MEMe810 (ZEO I

MO:15), using as a matrix rAVZ. The resulting fragment was cleaned and split by Vatni and Nipai! and cloned into the split Vatni-Nip and the purified pAV4 plasmid. The resulting plasmid, pAV5, encodes a molecule

BAV4Z3-34.

All methods of PCR and cloning were essentially performed as described in (Zatogook, ., Egiizsp, E.E. "5 Mapiai5, T. 1989 "MoiIesshag siopipd: a Iarogayugu tapiai!". 2pa. Ed. Soia Zriipd Nagbog I arogayugu, M.M.).

For all methods of cloning, E. soy K-12 cells, strain Tor-10 EE" (Zigaiadepe, OA) were used.

The rET28p vector was obtained from Momadep, O5A. All primers were synthesized at OMA Thesppoiod, Oeptagk.

Expression and purification of PAV43--34. Protein PAV43--34, encoded by pAV5, was expressed in E. soy cells

VI 21-Soid (Momadep) as described by the manufacturers of the rET28r system (Momadep).

The expressed BAV43--34 protein was purified to more than 8595 purity by washing the inclusion bodies followed by cation-exchange chromatography in the presence of 6M urea using an automated workstation for the purification of VioSai (RegZeriime Viosuziet5, BA). The urea was then removed by stepwise dialysis against a solution containing decreasing amounts of urea.

The final buffer was 10 mM Ttgiz, pH 8.5.

Immunization research. Mice transgenic for human APP (Aldheimer's disease precursor protein) were used for the study. These mice, called TOAEMOd8", express a mutant form of APP, which leads to high concentrations of Ар-40 and Ар-42 in the brain of mice.

Mice (8-10 mice per group) were immunized with either Areia-42 (5EO IU MO:2, residues 673-714, synthesized using a standard method from Etos) or variant PAV4A43---34 (construction 34 in the table in example 1 , obtained by the recombinant method) four times with two-week intervals. Doses were 100 mg for

Ar or 50 mg for PAV43--34. Blood was taken from mice on day 43 (after three injections) and after day 52 (after four injections), and to determine the level of specific antibody titers against Ar-42 using a direct

BSIZA AD-42 used serum.

The following table shows the mean relative antibody titers against Abeia-42.

As it becomes clear, the antibody titers obtained during immunization with the variant AD pAV43--34 are approximately 4 times and 7.5 times higher after 3 and 4 immunizations, respectively, than the titers obtained with the use of unchanged wild-type AD-42 as an immunogen . This fact can be further appreciated in perspective if we take into account the fact that the amount of variant used for immunization is only 50905 of the amount of wild-type sequence used for immunization.

Example C

Synthesis of AD copolymer peptide vaccine using an activated polyhydroxypolymer as a structuring agent.

Introduction. A traditional conjugated vaccine consists of a (poly)peptide covalently bound to a carrier protein. The peptide contains B-cell epitope(s) and the carrier protein presents T-helper epitopes. However, most carrier proteins are normally unsuitable as sources of T-helper epitopes, as only a small fraction of the entire sequence contains suitable T-helper epitopes. Such epitopes can be identified and synthesized as peptides, for example, 12-15 amino acids each. If these peptides are covalently bound to peptides that contain

B-cell epitopes, for example, with the help of a polyvalent activated polyhydroxypolymer, it is possible to obtain a vaccine molecule that contains only significant areas. In the future, it is possible to obtain a vaccine conjugate that contains an optimized ratio between B-cell and T-cell epitopes.

Synthesis of activated polyhydroxypolymer. Polyhydroxy polymers such as dextran, starch, agarose, etc. can be activated by 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride) or by homogeneous synthesis (dextran) dissolved in M-methylpyrrolidone (MMP) or by heterogeneous synthesis (starch, agarose, structured dextran), for example, in acetone.

Under dry conditions, 225 ml of dry M-methylpyrrolidone ( The flask was placed in an oil bath with a temperature of 60"C with magnetic stirring. The temperature was raised to 92"C for a period of more than 20 minutes. When the dextran was dissolved, the flask was immediately removed from the oil bath, and the temperature in the bath was lowered to 40"C. The flask was again placed in the oil bath, still with magnetic stirring, and tresyl chloride (2.7b4ml, 25mol) was added dropwise. After 15 minutes dry pyridine (anhydrous, 2.020 ml, 25 mol) was added dropwise. The flask was removed from the oil bath and stirred for 1 hour at room temperature. The product (dextran, activated with trezil, TAU) was precipitated in 1200 ml of cold ethanol (99.995). The supernatant was drained and the sediment was collected in 50 ml polypropylene tubes. in a centrifuge at 2000 rpm. The precipitate was dissolved in 50 ml of 0.595 acetic acid, dialyzed 2 times against 5000 ml

0.595 acetic acid and freeze-dried. TAYUO can be stored at -20"С in the form of freeze-dried powder.

An insoluble polyhydroxypolymer, such as agarose or structured dextran, can be activated with trezil by preparing a suspension of the polyhydroxypolymer, for example, in acetone, and carrying out the synthesis as a solid-phase synthesis. The activated polyhydroxy polymer can be collected by filtration. Appropriate methods are published, for example, in (Miiz5op K apa Mozrasp K (1987), Meshpoaz ip

Eplutiodu 135, p. 67, and in Neppapezop ST ei ai. (1992), ip "Ittobiiigea Ayipyu Gidapa Tesppidce5", Asadetis

Rgezv, Ips., p.87|.

Synthesis of copolymer peptide vaccines A Veia. TAO (10 mg) was dissolved in 100 μl of HgO and 1000 μl of carbonate buffer, pH 9.6, containing 5 mg of AD-42 (5BEO IU MO:2, residues 673-714), 2.5 mg of P2 (ZEO IU

MO:4) and added 2.5 mg of RZO (ZEO IYUO MO:6). All peptides, Ar-42, P2 and RZO, contained protected lysine groups: they were in the form of lysine groups protected by 1-(4,4-dimethyl-2,6b-dioxocyclohex-1-ylidene)ethyl (RAE). Peptides were obtained by the standard method from Htos, where the standard Etos-I uz(Vos)-OH was replaced by

Ethos-I uv(Oae)-OH (obtained in Momabiospet, cat. Me04-12-1121), i.e. the amino group of lysine was protected by Oae instead of Bos.

The pH value was measured and adjusted to 9.6 using 1M HCl. After 2.5 hours at room temperature, hydrazine was added from the 8095 solution to the final hydrazine concentration of 895, the solution was incubated for

REFERENCE at room temperature, and immediately afterwards freeze-dried. The lyophilized product was dissolved in Neo and dialyzed extensively against HgO before final lyophilization.

The ratio between B-cell epitopes (AD) and T-helper epitopes (P2 and RZO) in the final product can be varied by applying different concentrations of these peptides at the synthesis stage. Moreover, the final product can be labeled with, for example, mannose (as a target for conjugation with APC) by adding aminated mannose to the carbonate buffer at the synthesis stage.

If an insoluble activated polyhydroxypolymer is used to bind peptides containing

B-cell epitope and T-helper epitopes, binding to the polymer can be carried out as a solid-phase synthesis, and the final product is collected and purified by washing and filtration.

As indicated in the main description, the approach described herein for the production of a peptide-based vaccine can be applied to any other polypeptide antigen where it is convenient to obtain a pure synthetic peptide vaccine, and where the polypeptide antigen in question provides sufficient immunogenicity in a single peptides

Example 4

Synthetic copolymer peptide vaccines

TAO (10 mg) was dissolved in 100 μl HgO and 1000 μl carbonate buffer, pH 9.6, containing 1-5 mg of the peptide

A (any immunogenic peptide of interest!), added 1-5 mg of P2 (epitope P2 of diphtheria toxoid) and 1-5 mg of RZO (epitope of RZO of diphtheria toxoid). The pH value was measured and adjusted to 9.6 using 0.1M CARRIER.

After 2.5 hours at room temperature, the solution was then immediately freeze-dried.

The freeze-dried product was dissolved in HgO and extensively dialyzed against HgO or desalted by column gel filtration prior to final freeze-drying. In case of presence of lysine in the peptide sequence, the amine in the side chain of lysine should be protected with the help of BOaee with the use of Etos-I derivative uv(Oae)-OH in the synthesis (proposed by Sgedogii5 apa Tpeizep 2001). After binding, hydrazine was added from the 8095 solution to the final concentration of hydrazine in the range of 1-2095, and the solution was incubated for another ZOhv. at room temperature, immediately followed by lyophilization, and extensively dialyzed against HgO or desalted by column gel filtration prior to final lyophilization. The principle is schematically outlined in Figure 2.

As "peptide A", the authors of this invention used such immunogens as immunogens with a short C-terminal fragment of the O5rs protein from Vogtelia rigdodopegi, and as "peptide B" - with the epitope of diphtheria toxoid (P2 or P30). The results of immunization studies with this antigen revealed that only the immunogen according to the invention, which includes the O5rs fragment and a foreign diphtheria epitope corresponding to the haplotype

MHC of vaccinated mice was able to induce in these mice antibodies that react with O5rs. In contrast, a molecule containing only the O5rsC peptide was unable to induce antibody production, and the same was true for a mixture of 2 immunogens, one containing O5rs and the other an epitope. Therefore, it was concluded that inclusion in the same polyhydroxypolymer carrier is preferable, if not indispensable, for the induction of antibody production against a short peptide hapten similar to O5rs.

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LIST OF SEQUENCES er Rezvtekha DV "1202" New method of down-regulation of amiloykh "1302 P1014ВСИ "IA "1dih "160516 "Lo" Rali Me "aI1 "112 2313 "o" DNA "RIZ" Noto zarien: "220" "1 SO au" (U. (2313) "0 "RR" different properties "2222 42098). (2159) hoo3" nucleotides encoding the transmembrane region cha "2212" different properties ko (2015). -190 "gu" "2217 different properties of "Zh" (2016) (2144) "о" Arega 42/43 220" "2212 different properties of "ah" (2014). 42142) kg232 Abe 42/43 "оо" vshoska ss Che Ye dse skd sec sku sb des de kosh zsu oso sv V

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