UA79426C2 - Combined adjuvant compositions - Google Patents

Abstract

The use of an aminoalkyl glucosamine phosphate compound, or a derivative or analog thereof, in combination with a cytokine or lymphokine such as granulocyte macrophage colony stimulating factor or interleukin-12, is useful as an adjuvant combination in an antigenic composition to enhance the immune response in a vertebrate host to a selected antigen.

Description

Description of the invention

The present invention relates to the use of an aminoalkyl-leucosamine phosphate compound, or its derivative, or 2 analogs in combination with a cytokine or a lymphokine, in particular, a granulocyte-macrophage colony-stimulating factor or interleukin-12, as a form (composition) of an adjuvant in an antigenic or immunogenic composition for strengthening immune response of the vertebrate host to the selected antigen.

The immune system uses various mechanisms to fight pathogens. However, not all of these mechanisms are necessarily activated after immunization. Protective immunity that is induced by immunization depends on the ability of the immunogenic composition to induce a suitable immune response to confront the pathogen or eliminate the pathogen. Depending on the pathogen, this may require a cell-mediated and/or humoral immune response.

The existing paradigm regarding the role of helper T cells in the immune response is that T cells can be divided into subpopulations based on the cytokines they produce, and that the individual cytokine profile 12 observed in these cells determines their function. This model of T cells contains two main subpopulations: TN-1 cells that produce interleukin-2 (II -2) and gamma interferon, which increase both cellular and humoral (in the form of antibodies) immune responses; and TH-2 cells that produce interleukin-4, interleukin-5, and interleukin-10 (11/-4, 1/-5, and 1Й/-10, respectively), which increase humoral immune responses (reference 1 in bibliography) . 20 It is often desirable to enhance the immunogenic efficacy of an antigen to elicit a stronger immune response from the immunized organism and to enhance host resistance to the antigen-bearing agent. In some situations, a shift in the immune response from a predominantly humoral (TN-2) response to a more balanced cellular (TN-1) and humoral (TN-2) response is desirable.

The cellular reaction includes the generation of the СО8-СТИ reaction (the reaction of СОвиcytotoxic T-lymphocytes). p 25 Such a reaction is desirable for the development of immunogenic compositions against intracellular pathogens. Protection against various pathogens requires strong mucosal responses, high serum titers, induction of STIs, and strong cellular responses. These responses were not provided by most antigen preparations, including commonly accepted subunit immunogenic compositions. Human immunodeficiency virus (HIV) is among such pathogens. , 30 Thus, there is a need to develop forms of antigenic compositions that are able to generate both humoral, Ha, and cellular immune responses of the vertebrate host.

Thus, the purpose of this invention is the use of combined compositions (forms) of adjuvants in antigenic compositions that contain an aminoalkyl leucosamine phosphate compound (ACP), or its derivative or analog, Ge»! combined with a cytokine or lymphokine, in particular, granulocyte-macrophage colony-stimulating factor 3o (OM-SZE) or interleukin-12 (1/-12) or an agonist or antagonist of the specified cytokine or lymphokine. in

In particular, AsP is 2-KK)-Z-tetradecanoyloxytetradecanoylamino|ethyl-2-deoxy-4-O-phosphono-3-0O-(К)-З-tetradecanoyloxytetradecanoyl-2-(К)-З-tetradecanoyloxytetradecanoylamino|- VD -O-glucopyranoside, which is known as 529 (formerly known as KS529). C7C 70 An adjuvant is a substance that enhances the immune response when administered together with an immunogen or antigen. c The composition of the adjuvant of this invention is administered together with the selected antigen in an antigenic or immunogenic composition. Antigenic compositions of this invention enhance the immune response of the vertebrate host to this selected antigen. The selected antigen may be a polypeptide, peptide, or fragment derived from (1) a pathogenic virus, bacterium, fungus, or parasite, or |2) from a cancer cell or tumor cell, or |C| from an allergen so that it interferes with the production of CE to suppress allergic reactions to that allergen, or (4) from an amyloid precursor protein to prevent or treat a disease characterized by the deposition of (Ce) amyloid in a vertebrate host. In one embodiment of the present invention, the selected antigen is derived from HIV. The selected HIV antigen may be an HIV protein, polypeptide, peptide or fragment derived from said protein. In a particular embodiment of the present invention, the HIV antigen is a specific peptide. In another embodiment of the present invention, the selected antigen is β-amyloid peptide (also called β-peptide), which is an internal fragment

Ф protein-precursor of amyloid (APP), consisting of 39-43 amino acids, which is formed by the processing of APP by p- and y-secretase enzymes.

ACP can be present in the form of an aqueous solution or in the form of a stabilized oil-in-water emulsion (stable emulsion or ZE). In a preferred embodiment of this invention, the oil-in-water emulsion contains squalene, glycerol, and phosphatidylcholine. In the ZE-form, ACP is mixed with a cytokine or lymphokine to obtain (F) an antigenic composition before administration. There is no need for cytokine or lymphokine to be included in this emulsion. In d) the best version of this invention, the ACP is in the ZE form. The antigenic composition may additionally contain a diluent or carrier. The present invention also relates to methods for increasing the antigenic capacity of a composition containing a selected antigen |1) from a pathogenic virus, bacterium, fungus or parasite to induce an immune response in a vertebrate host, or |2)| from a cancer antigen or tumor cell to induce a therapeutic or prophylactic anticancer effect in a vertebrate host, or |C) from an allergen to prevent the production of IDEs to inhibit allergic responses to that allergen, or |4) from a molecule or part thereof representing a molecule or a portion thereof that is produced by the host (autologous molecule) in an undesirable manner, in an undesirable amount or location, to reduce such an undesirable effect, by including an effective adjuvant (immunostimulating) amount of a cytokine or lymphokine combination, in particular ACP with COM-C5E or II -12, or an agonist or antagonist of the specified cytokine or lymphokine.

Further, the present invention relates to methods of increasing the ability of an antigenic composition containing an antigen selected from a pathogenic virus, bacterium, fungus or parasite to induce cytotoxic T-lymphocytes in a vertebrate host by including an effective adjuvant (immunostimulating) amount of a cytokine or lymphokine combination, in particular, ACP with (3M-C5ZE or II-12, or an agonist or antagonist of the specified cytokine or lymphokine.

Figure 1 depicts the geometric mean titers of antibodies to Aβ1-42-peptide in transgenic mice immunized as follows: Group 1-Aβ1-42-peptide plus RVZ (not shown); Group 2-Ar1-42-peptide plus MRItm ZE 70 (squares); Group Z-Ar1-42-peptide plus MR tm BE and ZM-C5E (triangles); Group 4-Ar 1-42-peptide plus 529 ZE and

OM-S5BE (inverted triangles).

Figure 2 depicts the total cortical levels of AR in transgenic mice immunized with the four groups described for Figure 1.

Figure C depicts cortical levels of Aβ1-42 peptide in transgenic mice immunized with the four groups described for Figure 1.

Figure 4 depicts frontal cortex amyloid burden in transgenic mice immunized with the four groups described for Figure 1.

Figure 5 depicts the neurite burden of the frontal cortex in transgenic mice immunized with the four groups described for Figure 1.

Figure b depicts levels of retrosplenial cortical astrocytosis in transgenic mice immunized with the four groups described for Figure 1.

Figure 7 shows the geometric mean antibody titers of CA(E9M)-MZvo vr-peptide of HIV-specific IDS in serum in two groups of cynomolgus macaques (four animals per group). Group 1 animals were immunized intranasally only with C4(EZM)-MZvovr-peptide. Group 2 animals were immunized intramuscularly with He CA(E9M)-MZvo vr-peptide prepared from 529 ZE and 5M-SzR. Arrows indicate immunizations at weeks 0.4, 8, 18 and 23.

Figure 8 depicts the geometric mean antibody titers in the cervical-vaginal lavage samples of the animals described for Figure 7.

Figure 9 depicts the geometric mean antibody titers in nasal wash samples from animals described for (Ce)

Figures 7. sch

Adjuvants, cytokines and lymphokines are immunomodulatory compounds that have the ability to enhance the development and control the development and profile of immune reactions against various antigens, which are themselves weakly immunogenic. Appropriate selection of adjuvants, cytokines, and lymphokines can induce good humoral and cellular immune responses that would not develop in the absence of the adjuvant, cytokine, or lymphokine. In particular, adjuvants, cytokines and lymphokines have significant effects in enhancing the immune response to subunit and peptide antigens in immunogenic compositions. Their stimulatory activity is also useful for the development of antigen-specific immune reactions directed against protein antigens. For various antigens requiring strong mucosal responses, high serum titers, STI induction, and strong cellular responses, adjuvant and cytokine/lymphokine combinations provide stimuli not provided by most antigen preparations.

Numerous studies have evaluated various forms of adjuvants in animal models, but currently alum-c (aluminum hydroxide or aluminum phosphate) is the only adjuvant licensed for widespread use in humans. Another adjuvant is Stimulontm C)5-21 (Zythioptm 05-21) (Apiidepisv Ips., Egatipopat, MA (21). One » group of adjuvants, stable emulsions consisting of various combinations of oil or oil-in- water, has attracted much attention due to their immunopotentiating ability. These forms usually consist of various combinations of metabolized or inert oils that stabilize or deposit the antigen at the site of infection. One such adjuvant is Freund's incomplete adjuvant (IGRA), containing contains mineral oil, water, and an emulsifying agent. Freund's complete adjuvant (CEA) is IRA plus heat-killed mycobacteria. A particular concern with the use of these types of adjuvants has been injection site irritation, which often is the result of mononuclear cell infiltration that causes granulomatous lesions Thus, other compounds and 7 50 forms are being investigated as potential adjuvants.

One group of such compounds are aminoalkyl leucosamine phosphate compounds (ACP), which are described in (Patent

I) United States Mob6113918), e.g., column 2, line 14 - column 3, line 38, incorporated herein by reference (|Z. AOsR have an aminoalkyl (aglycone) group which is linked by a glycosidic bond to a 2-deoxy -2-amino-20-O-glucopyranose (glucosamine) to form the core structure. Additional substituents 22 include phosphorylation of carbon 4 or 6 in the glucosamine ring and three 3-alkanyloxyalkanoyl

GF) residues.

One such AOSP is a compound designated as 529 (whose full chemical name is o 2-KK)-3-tetradecanoyloxytetRadecanoylamino|ethyl-2-deoxy-4-O-phosphono-3-0O-(K)-3-tetradecanoyloxytetrad ecanoyl|-2-((K)-Z-tetradecanoyloxytetradecanoylamino!-pD-O-glucopyranoside), produced by Sogiha (Natiyop, 60 Mt.

Sogiha has also produced a metabolised form of an oil-in-water emulsion, which when combined with 529 results in a stabilized emulsion called 529 ZE. This stabilized emulsion is obtained by microfluidizing 529 with squalene oil, glycerin and phosphatidylcholine. The currently existing form is microfluidized CMR-grade emulsion Emulsions containing 195 oils (although other concentrations may be used) are described in the experiments below.

529 5E resulted in indistinguishable macroscopic tissue pathology when administered subcutaneously to Wair/c or Zm/izz-Uerzieg mice. A stabilized emulsion containing the same components but without 529 was also prepared for comparative purposes. Specifically, subcutaneous immunization with a 39 amino acid cysteine-deleted version (-Suv) of the 40 amino acid HIV peptide TIBRIOMMCA) (which lacks the cysteine residue at amino acid 17 of the 40 amino acid peptide (nC3)) or Ar 1-42 (an internal fragment with 42 APP amino acids), which were prepared with a combination of adjuvants 529 5E and OM-C5BE, did not lead to noticeable inflammation, redness, swelling or compaction.

The scope of this invention also includes derivatives and analogues of 529 and other AORs. Such compounds include, but are not limited to, the compounds described in (United States Patent Mob113918 (3)). 70 The inclusion of cytokines and lymphokines in immunogenic compositions showed promise in terms of expanding and strengthening the potential of immunogenic compositions I4)1. It was shown that the cytokine interleukin-12 (1-12) induces and enhances cell-mediated immunity through a shift in the expansion of the subpopulation of T-helper cells towards the profile of Tp1 cytokines (ie, towards the IdbC2 subclass in the mouse model) (5-7). Recombinant murine 1-12 has been shown to enhance the profile of Tp1-dominant immune responses in mice (4).

I--12 is produced by various antigen-presenting cells, mainly macrophages and monocytes. This is a critical element in the induction of TNI1 cells from "untrained" (naïve) T cells. It was shown that production

I--12 or the ability to respond to it is critical in the development of protective TNI1-like responses, for example, during parasitic infections, especially leishmaniasis |8), as well as the enhancement of the cell-mediated immune response to an antigen from a pathogenic bacterium or a pathogenic virus (9) or from a cancer cell (101Ї1. The effects of 11-12 are mediated to a large extent by interferon-gamma, which is produced by killer cells (MC) and T-helper cells. Interferon-gamma is critical for the induction of Idbs2a-antibodies to dependent protein antigens |11) and

IdoZ-reactions to T-independent antigens (|121). 1-12, originally called natural killer cell-stimulating factorb, is a heterodimeric cytokine (13). The expression and isolation of the I/-12 protein in recombinant host cells is described in the published International Patent Application UMO 90/05147 (141). p

Another cytokine that has the potential to be used as an adjuvant is ZM-C5E. (ZM-SZE is a special type of colony-stimulating factor (C5E). CE factors are a family of lymphokines that induce the differentiation of progenitor cells found in the bone marrow into specific types of mature blood cells. As described in (United States Patent No. 5078996 |15Y| ) which is incorporated herein by reference, ZM-C5E activates macrophages or monocyte progenitors to mediate nonspecific destruction of cancer cells (He)

From activity. The nucleotide sequence encoding the human ZM-SZE gene was described |15). Plasmid containing

The OM-C5E cDNA was transformed into E. soya and deposited in the American Type Culture Collection (ATSC), 10801 Opimegeiu Voshemaga, Mapazzaz, MA 20110-2209, under accession number 39900). As described in (US Pat. Vol

Mo5229496 |161), which is hereby incorporated herein by reference, the ZM-C5E gene was also inserted into a yeast expression plasmid and deposited in ATCC No. 531571. In addition, as described in US Pat. hereby incorporated by reference, the DNA sequence encoding OM-C5E, which has its glycosylation sites removed, has been deposited in ATCC Accession No. 672311.

CM-C5E has been shown to positively regulate protein molecules on antigen-presenting cells, which are known to enhance immune responses (18) and affect Id secretion in mouse B-cells purified by sorting (19). OM-C5E was also described as an adjuvant for immunogenic compositions (201.

Other cytokines or lymphokines have been shown to have immunomodulatory activity, including, but not limited to, interleukin-1-alpha, 1-beta, 2, 4, 5, 6, 7, 8, 10, 13, 14, 15, 16, 17 and 18, and interferons-alpha, beta and gamma, granulocyte colony-stimulating factor and tumor necrosis factors alpha "» and beta.

Important in connection with the systemic introduction of any cytokine or lymphokine are the biological consequences associated with the activity of cytokines or lymphokines. In addition, the actions of cytokines or lymphokines associated with the development of antigen-specific immune responses should be enhanced if local concentrations of cytokines or lymphokines are maintained. o Evaluation of combinations of 3-O-deacylated monophosphoryl lipid A or monophosphoryl lipid A with "iz" OM-S5BE or 1I-12 was carried out; observed an increase in various parameters of the immune response (211.

The invention described herein demonstrates that by combining an antigen, a selected cytokine or lymphokine as an adjuvant and a second ACP adjuvant (preferably in a stable metabolizable emulsion), immune

Ф reactions specific for this antigen are enhanced.

Antigens selected for inclusion in the antigen compositions of this invention contain peptides or polypeptides derived from proteins, proteins, as well as fragments of any of the following components: saccharides, proteins, poly- or oligosaccharides or other macromolecular components. As used herein, "peptide" refers to a sequence of at least three amino acids and contains at least one antigenic determinant or (F, epitope), while "polypeptide" refers to a longer molecule than a peptide, but does not constitute a full-length protein. Such peptides, polypeptides, or proteins may be conjugated to an unrelated protein, such as tetanus toxoid or diphtheria toxoid.As used herein, a "fragment" includes a portion that is less than all of said component, such as a saccharide, protein, poly- or oligonucleotide or other macromolecular component.In the case of HIV, the antigenic compositions of the present invention additionally contain full-length HIV proteins.

The present invention is first illustrated on the example of a model system using peptide antigens derived from HIV. These peptides are described in US Patent No. 5013548 |22| and Mo5019387 (231), incorporated by reference, or produced from the peptides described therein, and these descriptions are summarized below. These peptides contain 65 amino acid sequences corresponding to the region of the HIV envelope protein against which neutralizing antibodies and T-cell responses are elicited.

HIV is a human retrovirus that causes acquired immunodeficiency syndrome (AIDS). HIV infects

T-lymphocytes of the immune system by attaching the glycopoietin of its outer shell to the CO4 (T4) molecule on the surface of T-lymphocytes, thus using this CO4 (T4) molecule as a receptor for entering T cells and

Infecting them. Attempts to induce a protective immune response specific to HIV infection through immunization have had very limited success. Nowadays, a number of approaches are used in an attempt to determine an effective and protective strategy for the development of immunogenic compositions. They contain attenuated and recombinant bacterial vectors that express antigenic epitopes from HIV |24), recombinant adenovirus (25) or vaccinia virus vectors (26), DNA immunization and synthetic peptides containing various T- and B- cellular epitopes of HIV (281. 70 It has been shown that the glycoprotein dr120 of the outer envelope of HIV is capable of inducing neutralizing antibodies in humans. It has been shown that the recombinant PB1 protein, covering approximately one third of the complete molecule of or120, contains a part of the envelope protein that induces the formation However, studies in chimpanzees have shown that neither intact odr120 nor RVI1 is capable of inducing the production of high titers of neutralizing antibodies.

Short peptides were synthesized by conventional methods, and these peptides corresponded to the antigenic determinants of dr120 and generated a reaction in the form of antibodies against dr120, which neutralized this virus and induced T-helper and STI reactions against this virus.

One such peptide is the HIV-Itp peptide, which contains a C4-MZ multi-epitope called TISRIOMMA) (Suz) and a cysteine-deleted variant TISRIOMMA(A) (-Suv). These peptides contain TI, T cells and B-epitopes, but do not induce antibodies that prevent CO4 binding. It was previously demonstrated that these C4L/Z-peptides

HIV is a promising candidate for the induction of immune responses when administered with SEPA or

CEA-like adjuvants (29-34). These peptides contain epitopes that have been previously shown to induce srp4vTp cell responses in both mice and humans, and they contain both a major neutralizing determinant and a site that is recognized by CO8-STI as in Vair/c mice. , as well as in people who are NI A V7zh. Recently, it has been shown that the 39-amino-acid c g peptide has both immunogenicity and safety in HIV-infected patients (281.

TIZRIOMM(A) (ySuvz) has the following sequence of 40 amino acids: i) pt eh kot t l Хx Zl ПЕ u tat y He kiv mo vag si chit tk pa still tv zhni). z la la puh and zh put (12) sch

TIBRIOMIM(A) (-Suvz) was synthesized without cysteine at position 17 and it has the following sequence of 39 amino acids: Ge! vd "a " " du This cysteine residue is localized outside of functional epitopes that are recognized by TP cells, STI. with or B cells. Other HIV peptides from different regions of the viral genome are described in (US Patent No. 5861243, ZBI, US Patent No. 5932218, US Patent No. 5939074, US Patent No. 5993819, US Patent No. 6037135, US Patent No. 6037135, Published European Patent Application No. 71947 I40) and US Patent No. 6,024,965 |411), which are also incorporated by reference.

A peptide conjugate of 28 amino acids called 5T1/p11cS was also used. This conjugate consists of - 395 ZIM-epu-derived T-helper peptide of 16 amino acids, called 5-1, conjugated with SbZad peptide

ZIM-tas 251 of 12 amino acids (amino acids 179-190 Sad), named p11C |42). Peptide p11cS in tetrameric (Ce) form demonstrated STI activity in 5IM-tas infected rhesus macaques Maty-A"01 (43). Peptide 1" conjugate ZT1-p11C has the following amino acid sequence:

F KU Vo Chai Zhuk horror 8ih in Sue Tykh iho here ashrt tiv Asha ibh to zam (BNO Ho zhoa3),

A peptide conjugate of 39 amino acids, named C4-MZvo br (44), was also used. District C4 of this

GF) peptide conjugate (16 amino acids) produced from the fourth constant region of the HIV-1 envelope protein and represents a universal T-helper epitope. The MZ3 part of this peptide (23 amino acids) is derived from the third hypervariable region of the HIV-1 envelope protein and is a critical neutralizing determinant.

The Ca-MUZ conjugate in vr has the following amino acid sequence: 60 b5

There was a long time ago

Be Via Moi Tuk Aza ish aku zt ia At we shchi NU I MIZH ate dak t31yu Fu pt "u yo aa To Tuk 15 lk aku (what Shaw ate,

The HIV antigen can be a protein, polypeptide, peptide or fragment derived from said protein. 70 This protein can be a glycoprotein such as orp41, dr120 or dr1460. Alternatively, this protein may be a protein encoded by genes such as dad, roi, myi, hem, mrg, fai, pei or epu. Peptides produced from such proteins will contain at least one antigenic determinant (epitope) at least six amino acids long.

The immune response to the HIV peptide can be enhanced by covalent binding (conjugation) of the peptide with a 79 molecule of a pharmaceutically acceptable carrier. Examples of suitable carrier molecules include tetanus toxoid, diphtheria toxoid, fissurella hemocyanin, and other peptides corresponding to T-cell epitopes of the HIV glycoprotein dr120.

Currently, it is assumed that a successful strategy for immunization against HIV will require the induction of mucosal immunity to HIV as well as a good StT response. In a recent study in mice using the multiepitope peptide TISRIOMM(A) and the mucosal adjuvant, cholera toxin, it was shown that intranasal immunization induced neutralizing serum antibodies to IdsC1 (45). The next study, also using peptides of the MZ loop of HIV, showed the induction of synthesized mucosal

IdA antibodies and strong cell-mediated reactions, including peptide-specific STIs. (46). The functional role of high titers of systemic and neutralizing antibodies in HIV prevention or stabilization of HIV-infected individuals is unknown, although high titers of virus-specific antibody are thought to be important in preventing d) spread of the virus.

In a preferred embodiment of this invention, a stable oil-in-water emulsion containing 529 is prepared, which is then mixed with cytokines 1-12 or CM-C5R. The data presented below show that the combination of 529 plus

O-M-C5E leads to high titers of HIV-neutralizing serum antibodies. The combination of 529 5E and EM-C5E ise) induces high titers of antigen-specific IdS antibodies, including both IDS and IdS2a subclasses, in the vaginal pool of immunized female mice. Immunization of mice with TIZRIOMM(A) (-Suv)-peptide prepared from 529 ZE and

OM-C5BE induced a strong cellular immune response, as determined by enhanced antigen-specific cell proliferation and secretion into the culture of cytokines, as well as by the induction of peptide-specific STI reactions. Similar Ge! the results were observed when mice were immunized with Ар 1-42-peptide from APP with 529 ZE and ЗМ-С5БЕ.

Usually, the antigen/adjuvant form of 529 or 529 ZE, combined with ZM-C5E or 1I/-12 and the selected protein and peptide, induces high titers of antigen-specific and neutralizing virus antibodies, a significant shift in the ratio of subclasses (to the side of greater fractions of complement-fixing Ids-antibodies (in favor of Ids2a in mice), increased cytokine production and cell proliferation from mononuclear cells in response to stimulation with the ip myo antigen. These properties were not observed with antigen and 5E forms in the absence of 529, nor with

OM-S8BE or II-12, not without CM-S5E or II-12. The forms of this invention also induce good cellular responses, as defined by the induction of STI.

The advantage of 529 5E is that this form does not induce granulomatous accumulation and inflammation at the injection site; such injection site reactions are usually induced by water-in-oil or oil-in-water forms of adjuvants.

An experiment was conducted to compare the introduction of HIV peptide TISRIOMM(A) (-Suz) only with 529 5E or with 7 529 ZE plus 1-12 or 529 BE plus CM-C5E. (Te) In this experiment (Table 71, below), Wair/c mice immunized subcutaneously with the HIV peptide (TIZRIOMMA) (-Suv) prepared from 529 ZE induced peptide-specific serum titers of dO after only two injections. The titers of Ids1 and Ido2a subclasses were also induced. Inclusion of the second adjuvant, or ZM-SZE, or ka 20 | -12, increased the total titers of IDSS, as well as the titers of the subclass of IDSI. The addition of 1-12 increased the titers of the subclass I(d2a; the addition of ZM-C5E did not increase the titers of the subclass IdSg2a. In another experiment, as a measure of functional cell-mediated immunity, the ability of spleen cells of mice immunized with 529 5E or 529 5E plus 1-12 or 529 was evaluated 5E plus OM-C5E, prepared together with the multi-epitope peptide TIZRIOMMA) (-Suz), generate ViILu-specific STI reactions.

As shown in Table 2, spleen cells from mice immunized with each of the adjuvants showed low (PI) activity against target cells that were either unlabeled or transiently labeled with a foreign STI epitope

IV. VillIu-specific STI-mediated lysis of target cells was markedly enhanced by administration of 529 ZE di plus 1-12 compared with 529 5E alone, and was further enhanced by administration of 529 5E plus CM-C5E (Table 2). because in another variant, rhesus macaques were immunized with peptides 5T1-p11C or C4-MZdovr and IRA or 529 5E plus

OM-C5E (groups shown in table 15). The results of these analyzes are shown in Tables 16-22 and are now summarized.

The very form of the peptide ZT1-p11C is apparently well tolerated in all tested animals. However, significant injection site reactivities were noted with IRA adjuvant. In addition, possible minor adverse effects of the adjuvant form 529 5Е/3М-С5РЕ were observed immediately after the final immunization. Peptide form

5T1-p11C containing IRA was able to induce a strong p1!1C-specific cellular immune response in one of the two Mat-A"01-positive rhesus macaques tested. The 5T1-p11C peptide form containing 529 5BE/3M-C5E , was also able to induce a p11C-specific cellular immune response in one of the two tested

Mothers of A"01-positive rhesus macaques.

The C4-MZvovr peptide form, containing IRA, was able to generate the maximum titers of EHIBA antibodies in plasma in the range of 1:25600-1:102400 and titers of serum neutralizing antibodies against ZNiIMdo in and ZNIMao vr.

The C4-MZvo vr peptide form, which contains 529 ZE/5M-C5E, was able to generate maximum titers of EGISA antibodies in plasma in the range of 1:6400-1:12800 and low levels of neutralizing antibody reactions against ZNIMavov, but not against 70. ZNIMvovr.

With a small number of animals per group (two), it is difficult to draw specific conclusions. However, the level of immune response, both humoral and cellular, generated by both forms of peptides containing 529 5BEHZM-C5ZE was quantitatively lower than the immune response observed in animals administered IRA as an adjuvant. It should also be noted that IRA is not an approved ingredient in commercial human formulations. 7/5 In addition, there is some limited evidence that the functional properties and phenotype (ie, cytokine profiles) of responder cells may differ depending on the form of adjuvant used.

In yet another experiment, animals from a second species of primate, the cynomolgus macaque, were immunized with the peptide

C4-MZdovbr, which was modified by replacing glutamic acid at amino acid residue 9 with valine.

The resulting peptide conjugate, named C4A(E9M)-MZdoer, has the following sequence: 7

She aku shi la ate your yi1yi4 biu zha SCHI Mk sch da zhiyu There are people living here (80 to shchinb). at

Animals were immunized with CA(E9M)-MZvo vr peptide, either without adjuvant or with a combination of 529 ZE plus CM-C5E.

These results indicate that C4(E9M)-MZdobr peptide when administered by intramuscular injection in combination with 529 BE/ZM-C5E induces significantly higher titers of peptide-specific antibodies in serum than the same "ozo" alone. the amount of C4(E9M)-MZvebRg peptide administered intranasally without an adjuvant (Figures 7-9). The results of this experiment clearly demonstrate that HIV peptide immunogen when administered in combination with a suitable combination of adjuvants is capable of inducing systemic humoral immunity. "IS

Desirable immunogenic compositions for the prevention or treatment of diseases characterized by the deposition of amyloid (autologous molecule) in a vertebrate host, which contain combinations of adjuvants of this Me

According to the invention, they contain compositions containing parts of beta-amyloid precursor protein (APP). This M disease is called in different ways: Alzheimer's disease, amyloidosis, or amyloidogenic disease, p-amyloid peptide (also called Ar-peptide) is an internal fragment of APP with 39-43 amino acids, which is formed by processing of APP by pD- and y-secretase enzymes. The А1-42-peptide has the following "sequence: - so aerzhya m te vo Ma Mo sh ikh ih buh v tah Bi pl . shcha this is bad or Shk z il a Vr zha), she de shi Za

B schu mi tiv tiv Mia iya Yan Ti bu Bir na chii Tiz Ma shi z shp. -1 395 In some patients, amyloid deposition takes the form of aggregated A p-peptide. Unexpectedly, it was shown that the administration of the isolated A p-peptide induces an immune reaction against the A p-peptide component of amyloid deposition in the vertebrate host I47). Thus, the immunogenic compositions of the present invention contain combinations of the adjuvant of the present invention plus the Aβ peptide, as well as fragments, derivatives or modifications

Ar-peptide and antibodies to Ar-peptide or its fragments, derivatives or modifications. One such fragment of the Ар-peptide is a peptide of 28 amino acids with the following sequence (481:

Fal lzhi Zhe LEM Zhid Le ynsyu u ut Sha chnz shzhi iv

Fi he Bai t Z Shv viv bil Bar ay «id yl za Bal

With a shunt.

HF) Other Aβ peptide fragments of interest include, but are not limited to, amino acids 1-10, co 1-7, 1-6, 1-5, 3-7, 1-3 and 1-4 , which can be administered in unconjugated form or conjugated with a foreign protein.

A number of studies were conducted with A p1-42-peptide and various adjuvants. Next, the generalized results are presented.

In the first experiment, Zmizv-UUerzieg mice, immunized subcutaneously in the gluteal region with the A1-42-peptide, generated titers of peptide-specific antibodies Ids, IdsC1 and Ids2a, demonstrating that the A1-42-peptide is a viable vaccine antigen. Addition of ZM-C5E to 529 ZE and Ар1-42-peptide led to increased titers of antibodies Ids, Идс1 and dose in serum compared to recipients of 529 ZE and Ар1-42-peptide (see Table b5 3-8). Serum antibodies in individual mice receiving the combination of 529 ZE plus OM-C5BE increased more often and more rapidly than in individual mice receiving 529 ZE alone (data not shown). When repeating this first experiment with older mice

Zugizv-UMerwieg (aged 6-8 months instead of less than 3 months) observed results similar to those in Tables 3-8 (data not shown).

In the second experiment, Zm/izz-Uerzieg mice were immunized subcutaneously in the gluteal region with the A1-42 peptide and 529 ZE with varying amounts of CM-C5E. Endpoint titers increased in a dose-dependent manner as ZM-C5E increased (from 0.1 to 1 to 10 µg) (Table 9). Titers dO for all combinations 529 ZE plus

OM-C5E were higher than for groups receiving the other adjuvant, 05-21, alone or with ZM-C5RE. The titers of the YidDS1 subclass were also increased for different groups of 529 ZE plus ZM-C5E compared to the group receiving 7/0 529 ZE plus ZM-SZE in the first dose and only 529 ZE in the second dose (Table 10). Titers of the Id02a subclass also increased for the various 529 ZE plus ZM-C5E groups compared to the 529 5E alone group in a dose-dependent manner (Table 11).

In the third experiment, Zm/izz-Uervsieg mice were immunized subcutaneously in the gluteal region with A1-42-peptide and 529 ZE with varying amounts of CM-C5E or without varying amounts of CM-C5E. Endpoint titers of 75 µg increased for different groups of 529 5E plus ZM-C5E (0.5 to 2 to 5 to 10 μgHg), although not in a dose-dependent manner (Table 12). The titers of both IdsS1 and (d02a) subclasses also increased for different groups 529 5E plus

OM-S5BE in comparison with the group only 529 5E, although not depending on the dose (tables 13 (Pos11 and 14 Pdsg2a)).

A fourth experiment used transgenic mice expressing a variant form of the amyloid precursor protein (APP) that has a mutation at residue 717, with valine replaced by phenylalanine (49).

This mutation is associated with familial Alzheimer's disease in humans. These transgenic mice (called ROAPP mice) that progressively develop many of the pathological features of Alzheimer's disease, including Aβ deposition, neuritic plaques, and astrocytosis, and therefore serve as an animal model for human Alzheimer's disease.

In this fourth experiment, ROAPP mice were immunized subcutaneously with the A1i-42 peptide with or without various adjuvants and at the doses shown in Table A. In particular, group 1 mice received o

Ар1-42-peptide from MRI tm (Sogikha, Natiyop, MT) in stable emulsion form (ZE) as a positive control; mice of group 2 received A1-42-peptide with MRilm ZE plus murine ZM-C5E; mice of group C received

A1-42-peptide with 529 ZE plus mouse CM-C5ZE; group 4 mice received PB5 as a negative control. Groups 2 and

C showed a faster increase in anti-Ar1-42-antibody titers, as well as a higher maximum ke, titer than groups 1 or 4. si

However, the titers in groups 2 and 3 decreased to the equivalent titer of the positive titers of group 1 within 2-3 months (Figure A). Groups 1, 2, and C showed significant reductions in A D levels in the brain as measured by EGISA (Tables B-C and Figures B-C), lower amyloid loads (Table O and Figure 0), and (o) less neuritic dystrophy (Table E and Figure B), compared to the negative controls of group 4. Groups 27 and C had a significant reduction in astrocytosis compared to the positive controls of group 1 (Figure RE). -

Thus, the adjuvant (immunostimulating) properties of 529 ZE and ZM-C5E or II-12 are obviously synergistic when preparing a compatible composition.

The antigenic compositions of the present invention modulate the immune response by improving the response in the form of antibodies to the vertebrate host and cell-mediated immunity after the introduction of an antigenic composition containing an antigen selected from a pathogenic virus, bacterium, fungus or parasite and an adjuvant amount of ACP, such as 529 c (in aqueous form or in the form of a stable emulsion), combined with a cytokine or lymphokine, in particular ZM-SZE or :z» I--12. Other cytokines or lymphokines have been shown to have immunomodulatory activity, including but not limited to interleukin 1-alpha, 1-beta, 2, 4, 5,6, 7, 8, 10, 13, 14, 15, 16 , 17 and 18, interferons-alpha, beta and gamma, granulocyte colony-stimulating factor and tumor necrosis factor alpha and beta. - 395 Agonists or antagonists of certain cytokines or lymphokines are also within the scope of this invention.

As used herein, the term "agonist" means a molecule that enhances the activity of said cytokines or (Ce) lymphokines or functions in the same manner as said cytokines or lymphokines. An example of such an agonist is a 1" mimetic of said cytokines or lymphokines. As used herein, the term "antagonist" means a molecule that inhibits or prevents the activity of said cytokines or lymphokines. Examples of such antagonists are ko 50 soluble II-4 receptor and soluble TME receptor.

F As used herein, the term "effective adjuvant (immunostimulatory) amount" means a dose of a combination of adjuvants described herein that is suitable for inducing an increased immune response to a selected antigen in a vertebrate host, compared to a host receiving that selected antigen during in the absence of this combination of adjuvants. The specific dose will depend in part on the age, weight and medical condition of the host, as well as on the method of administration and the specific antigen. In the best case, this combination of adjuvants will

HF) use 529 in the range of 0.1-50 Ωkg per dose; in a better version, a range of 1-100 mcg 7 per dose is used. Appropriate doses are readily determined by those skilled in the art. Antigenic compositions of this invention can also be mixed with immunologically acceptable diluents or carriers in a conventional manner for the preparation of injectable liquid or suspension solutions. 60 Antigenic or immunogenic compositions of the present invention are administered to a human or non-human vertebrate by various routes, including, but not limited to, intranasal, oral, vaginal, rectal, parenteral, intradermal, transdermal | see, for example, MUMO International Application 98/20734, which is incorporated by reference), by intramuscular, intraperitoneal, subcutaneous, intravenous and intraarterial routes. The amount of the antigenic component or components of the antigenic composition will vary depending on the particular antigen, as well as the age, weight and medical condition of the host, as well as the method of administration. Again, appropriate doses of legso are determined by those skilled in the art.

Preferably, although not necessarily, the antigen and the combination of adjuvants are administered simultaneously. The number of doses and the dosing schedule for the antigenic composition are also readily determined by those skilled in the art. In some cases, the adjuvant properties of the combination of adjuvants can reduce the number of necessary doses or the time course of the drug administration regimen.

The combinations of adjuvants of the present invention are suitable for use in antigenic or immunogenic compositions containing a wide variety of antigens from a wide range of pathogenic microorganisms, including, but not limited to, antigens from viruses, bacteria, fungi or parasitic microorganisms that infect humans and 70 non-human vertebrates or antigens from a cancer cell or tumor cell. An antigen may contain peptides or polypeptides derived from proteins, as well as fragments of each of the following components: saccharides, proteins, poly- or oligonucleotides, cancer or tumor cells, allergens, autologous molecules (such as amyloid precursor protein), or other macromolecular components. In some cases, more antigens than one antigen may be included in the antigenic composition.

Preferred viral immunogenic compositions containing combinations of adjuvants of the present invention include compositions aimed at preventing and/or treating disease caused by, but not limited to, human immunodeficiency virus, simian immunodeficiency virus, respiratory syncytial virus, parainfluenza virus types 1-3, influenza virus, herpes simplex virus, human cytomegalovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human papillomavirus, poliovirus, rotavirus, caliciviruses,

Measles virus, mumps virus, rubella virus, adenovirus, rabies virus, canine distemper virus, rinderpest virus, human metapneumovirus, avian pneumovirus (formerly called turkey rhinotracheitis virus), Hendravirus, Nipavirus, coronavirus, parvovirus, infectious rhinotracheitis viruses, feline leukemia virus, infectious feline peritonitis virus, infectious bursal disease virus of birds, Newcastle disease virus of birds, Marek's disease virus, porcine respiratory and reproductive syndrome virus, equine arteritis virus and various encephalitis viruses.

Preferred bacterial immunogenic compositions containing combinations of adjuvants of the present invention include i) compositions aimed at the prevention and/or treatment of disease caused by, without limitation,

Naetoriyiz ipiiitseplae (both typical and atypical), Naetorpyi5 otpyv, Mogaheya sayagtpnaiv,

Зігеріососсивз рпецтопіає, Зігеріососсив руодепе5, Зігеріососсиз ада|асіаеє, Зперіососсив Таесаїів, «о зо Неїїсорасієг руог, Меївзепа тепіпуйідів, Меївзепа допогппоеае, Спіатудіа іаспотайв, Спіатуаїа рпештопіае, СПіатуадіа рвзіцасі, Вогдефейа репивзвів, АППоіїососсив оїйайів, ЗаіІтопейПйа (урпі, ЗаїІтопегІа с їурпітигішт, ЗаітопеПйа spoiegaeziiz, EvsPpegispia soii, ZPideMya, Mirgio sPpoIegae, Soguperasiegit «E dirpipegiae, Musobrasiegist (shbregsciovii, Musorasiegist amisht-Musobrasiegist izigaseishShiage sotrieh, Rgoieiv tigariyiv, Rgoiviz mzidagiv, Z(arpudosossiv atsgeipisos, e(ardepsiiv

With Heriozriga ipiegodap5, Voiteya rigodopegi, Ravietsgeia Naetoiuysa, Razieygeia tiosida, AsNporasiy5 r rieygorpeshtopiae and Musoriazta daizeriisit.

Preferred immunogenic compositions against fungal pathogens containing combinations of adjuvants of this invention include compositions aimed at preventing and/or treating disease caused by, but not limited to, Azregoiv5, Viaziotusez, Sapaida, Sossidiodez, Sguriosossiz, and Nizioriazta. "

Preferred immunogenic antiparasitic compositions containing adjuvant combinations of the present invention include compositions directed at preventing and/or treating a disease caused by, without limitation,

And eevptapia tayog, Avsagiv, Thispygiv, Siagaia, Zspiviozota, Stguriozrogidiyt, Tgispotopavz, Tochoriazta dopaii and ;" Rpeyshtosuviiv sagipiy.

Preferred immunogenic compositions for inducing a therapeutic or prophylactic anti-cancer effect in a vertebrate host containing combinations of the adjuvants of the present invention comprise compositions that employ a cancer antigen or a tumor-associated antigen, including, without limitation, a specific for prostate antigen, carcino-embryonic antigen, MOS-1, Neg2, CA-125 and MACE-3. and, Preferred immunogenic compositions for attenuating reactions to allergens in a vertebrate host, containing their combinations of adjuvants of this invention, contain compositions that contain an allergen or a fragment thereof.

Examples of such allergens are described in |(United States Patent Mo5830877 (51) and published International Patent Application Mo MUO99/51259 |52)|, which are hereby incorporated by reference), and include pollen, poisons

F insects, animal dander, fungal spores and drugs (such as penicillin). These immunogenic compositions prevent the production of IdE antibodies, a known cause of allergic reactions.

Preferred immunogenic compositions for attenuating reactions to autologous molecules in a vertebrate host containing combinations of adjuvants of this invention contain compositions containing an autologous molecule or a fragment thereof. Examples of such autologous molecules, in addition to the Aβ1-42 peptide described above, include iF) D-chain insulin involved in diabetes, molecule 517 involved in a disease such as gastroesophageal reflux, and antigens that negatively regulate (reduce) autoimmune reactions in diseases such as multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis. 60 In the case of HIV and 5IM (simian immunodeficiency virus), these antigenic compositions contain at least one protein, polypeptide, peptide or fragment derived from said protein. In some cases, multiple proteins, polypeptides, peptides and/or fragments of HIV or IM are included in the antigenic composition.

The combined forms of adjuvants of this invention are also suitable for inclusion of the adjuvant in polynucleotide immunogenic compositions (also known as DNA immunogenic compositions). Such immunogenic compositions may additionally contain auxiliary agents such as bupivicaine | see US Patent Mo5593972 |53), which is incorporated by reference).

For a better understanding of this invention, the following examples are presented. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the present invention.

Examples

Example 1

Materials and methods

The following materials and methods were used in the experiments reported in Examples 2-7 below.

Animals

Female Vair/s mice at the age of 7-9 weeks were purchased from TaiYisopis Ragt»v, Ips. (Segtapiomp, Moscow State University). Female 7/0 Zzhizv-UMerzieg mice aged 7-9 weeks were also purchased from TaiYisopis Ragtv, Ips. All mice were housed in facilities approved by the American Association for Laboratory Animal Care. Mice were acclimatized to the housing equipment for one week before the start of the studies.

Antigens

In the HIV experiments in Examples 2-3 below, two different synthetic peptides were used. 7/5 The sequence of the multi-epitope peptide NIM-1 (TIZRTOMMA) (-Suz) (also called here MM-10) is as follows:

He ate, ate, ate, ate, ate, ate, ate, ate, ate, ate, ate, ate, ate, ate, ate, ate, ate, ate, and ate three women.

Sho u l z Kok liv Ya Zhuk Chi i yshe a z ShZhnale

This peptide was described earlier |33, 34) and contains sequences from the HIV-1 d120 that induce CO4 7

Tr-cell responses in both mice and humans, the major neutralizing determinant and site recognized by CO8"

STI in Vair/s mice. This peptide was kindly provided by Dr. K. Zseagse |byke Opimegaiu, bigpat, MS). For analysis

100 for the purpose of comparison, a foreign peptide called SHV was used. This peptide corresponded to the STI epitope. in p. 22 of the UZ-loop Nim-1-u6 (Agu Siu Rgo Siu Agd AiIa Rpe Ma! Tig Pe (ZESO I MO:8)), and was purchased from Sepozuz (3

Vioyesppodiez Ips. |Pne UuUoodiapaz, TX). Peptides were solubilized in sterile water and diluted in suitable buffers or cell culture medium before use.

In experiments with amyloid of examples 4-6 and 8 below, a peptide called A1-42 was used. z The sequence of Ar 1-42 is as follows: i-th peace had bodies. Shi ze Yabo lyur or YaBu buh im chayi shiya shchin sch

Mil three ate ki shi Ya Kia yza ASHK ki z zh zh vla Be Z vyhla ma Ti Shu ila o uniya bu vid ka a yiv viv F z5 Ka t shiy, y-

This peptide has been described previously (47) and corresponds to an internal fragment of 42 amino acids of the amyloid precursor protein. Ар1-42 was obtained from EIAP РНаптацециисаиз (Zosh Zap Egapsizso, SA). The peptide was solubilized in sterile water and diluted in suitable buffers or cell culture medium before use. WITH

Adjuvants c All adjuvant preparations containing 529 were obtained from Sogih (Natiyop, MT). 529 5E was prepared in the form of a preliminary emulsion of the "oil in water" type (0.8-2.595 oil) based on squalene, which has a concentration of 529 in the range of 0-5 Ωkg/ml. Aluminum phosphate was prepared in our own laboratory. Complete Freund's adjuvant (CEA) and incomplete Freund's adjuvant (IGA) were purchased from Ohio State University, Ohio, MI. Peptides TI5RIOMMA) and adjuvants

Freund was emulsified in a 1:1 ratio using two paired syringes. Recombinantly 7 expressed murine I/-12 was obtained from Sepoys Ipzishe (Satregidde, MA). Recombinant mouse (se) OM-C5E was purchased from Viozoshgse Ipiegpaiippa! |Sataguio, SA) in the form of a lyophilized powder without a carrier.

Stimulant 5-21 was purchased from Apiidepisz Ips. |gatipoapat, MA). t Immunizations of 20 mice were immunized subcutaneously in the gluteal region in a total volume of O0.2ml, equally divided on each side of the gluteal region. Immunizations were performed at different time intervals, indicated below. Antigen c and cytokines were diluted in phosphate-buffered saline to suitable concentrations and prepared with adjuvants less than 16 hours before immunization under sterile conditions. Immunogenic compositions were mixed by careful shaking and stored at 4 9С. These forms were vortexed immediately before immunization.

GF) Serum analysis using solid-phase enzyme-linked immunosorbent assay 7 Animals were bled before the initial immunization and at the specified time points. Serum analysis was presented as the geometric mean of individual titers. For the analysis of antibodies specific for the HIV peptide and the distribution of subclasses, the peptide was suspended either in a carbonate buffer (15 mM Ma»CO», 35 mM Manso», bo p 9.6), or in РВ5, at a concentration of μg/ml and placed in 96-well microtiter plates tablets (Mips) in a volume of 100:1. After overnight incubation at 37 °C, the tablets were washed and blocked (0.195 gelatin/PB5) at room temperature for 2-4 hours. EGIZA tablets were washed with washing buffer (РВ5, 0.190

Twintm20) before adding serially diluted serum (PB5, 0.195 gelatin, 0.0595 Twin tm20, 0.0295 sodium azide 65). After a 4-hour incubation, the wells were washed and appropriate dilutions of biotinylated anti-isotype/subclass antibodies were added for overnight incubation at 42C. Wells were washed and incubated with streptavidin-conjugated horseradish peroxidase. After incubation, the wells were washed and AVT was detected. The wells were read at 405 nm. Titers were standardized using control sera.

The same protocol was used for the analysis of A p1-42-peptide-specific antibodies and subclass distribution, except that a concentration of 0.µg/ml per microtiter plate was used.

Obtaining cells

For proliferation assays and cytokine analysis, cells from the spleen of mice at the indicated time points were obtained. Suspensions of individual cells were obtained from pools of 3-5 mice. For the analysis of proliferation and cytokines, cells were suspended in round-bottomed 9b-well culture plates pre-coated with 7/0 overnight with HIV peptide antigens, control proteins or only KRMI-10 medium. Spleen cells were added at 5x10? of cells per well using a culture medium with 2x supplements.

Cell culture supernatants were collected from the wells in triplicate for cytokine analysis after three or six days after initiation of culture. Immediately after collecting the supernatants, the cultures were briefly treated with ZN-thymidine for 18-24 hours and collected for quantification of cell proliferation.

Example 2

Reciprocal end-point titers of anti-TIZRTIOMMA) (-Suv)-IdDO for general dos and subclasses

Reciprocal titers of endpoints of subclasses Id were measured from pooled serum (n-5 Vair/s) five weeks after the initial immunization, two weeks after the secondary immunization. Mice were immunized subcutaneously in the gluteal region with 25 μg of T1ZRIOMM(A) (-Suv) using a total of 0.2 ml, divided equally into two

O/Mml injections on each side at week 0 and week 3. 529 5E was diluted to make an emulsion containing 12590 squalene oil and 25 µg 529 per dose. BE is an oil-in-water emulsion-carrier consisting of squalene, glycerin and an emulsifying agent. Recombinant murine 1-12 was provided in an amount of 4 ng per mouse.

Recombinant murine ZM-C5ZE was administered at 25 μg per mouse. Results are shown in Table 1 with geometric mean titers plus standard errors for each group. with o 1111 purinnvvykhtyuyu 0

F from school -

Example From F

Analysis of STI in Vair/s mice v.

The protocols of example 2 were used to immunize mice. STI activity of spleen cells isolated from mice 14 days after secondary immunization was evaluated. 529 5E was prepared from 25 mcg of 529 ZE containing 1.2590 oil with 10 mcg of ZM-S5E or 40 Ong II -12 or without them, plus 25 mcg of TIZRIOMMA) (-Suz). "

For STI analysis. spleen cells were extracted from immunized mice 14 days after secondary immunization.

Actually, the previously described protocol was used. Briefly, cells from spleens that do not contain erythrocytes from three mice per group were pooled. Effector cells of the spleen (4x105 per ml) were re-stimulated in "» 24-well culture plates in a volume of 1.5-2 ml for seven days with 1 µg/ml of either the MM-10 peptide or the 10-mer peptide of the STI epitope. "PIV" or without NIM peptide. Both STI epitopes were restricted to H-209. Cultures were supplemented with 10U/ml of recombinant murine 1-2 (Viozoigse) during the last five days of cultivation. For 45 . with. . Bi. - analysis of the cytotoxic activity of P815 cells labeled with Sg" and briefly treated with 5 μg/ml of the peptide (PIV or

MIM-10) for four hours and added to cultured spleen effector cells. If the NIM peptide was not (Ce) used, then a series of target cells were not subjected to this short-term treatment. 1" three-fold dilutions of effector cells to target cells ("E:T") 30:1-1.1:1 were used. The percentage of STI activity was calculated as the percentage of chromium release using the equation ((specific release of 50 chromium-spontaneous release of chromium)(maximum release of chromium-spontaneous release of chromium))"100.

Ф Chromium release was assessed after a b-hour incubation period. The average spontaneous release of chromium was always less than 1595 of the maximum release. The results of the data from day 28 are shown in Table 2. zv o 0 penidmnoo | pelayadnv | Bezpeladu Z yu yav nova 48 v v vvrva 259 v 141 m 012 1312 3 b5 Example 4

Reciprocal end point titers of anti-Ar1-42-Ido for common Ids and subclasses

Zm/izv-UUervieg mice obtained by outbreeding were divided into groups of 10 mice each. Each group received ZOmkg of A1-42-peptide, which corresponds to the internal region of APP with a length of 42 amino acids.

The first group did not receive adjuvant; the second group received 5Omkg 529 ZE containing 2.595 oil; the third group received 50 μg of 529 ZE, containing 2.596 oils plus 1 Ωkg of M-SZE; the fourth group received 10 μg

OM-S5E; the fifth group received ZE containing 1.2595 oil; the sixth group received ZE containing 1.2590 oil plus 1 Ωkg of ZM-C5E; the seventh group received 5Omkg 05-21. Mice were immunized subcutaneously in the buttock region with a total volume of 0.2 ml, divided equally into each of two areas at the base of the tail/buttock region.

Immunizations were performed at week 0 and week 3. 70 Mice were bled on days 0, 20, 35, and 70. Serum was analyzed from individual mice. Reciprocal endpoint titers of anti-Ar1-42-peptide-dDO for total db and subclasses were measured in individual sera (n-10

Zmizz-UUerzieeg) at week 5 and at week 10. The results for the endpoints are shown in Tables C (week 5) and 4 (week 10). The results of the Ido1 subclass are shown in Tables 5 (week 5; no adjuvant or 05-21 groups were measured) and 6 (week 10). The results for the Ido2a subclass are shown in Tables 7 (week 5; 75 groups receiving no adjuvant or 05-21 were not measured) and 8 (week 10). o sch 2 o

Ф зо сх з

Ф з ш " ш ч г" з -

Fig. d) 50

Ф with o view 00010099 and ? tires bo Titers of end points of anti-Ar1-42-IdO week 5 o and yu

Example 5

Reciprocal titers of anti-Ar1-42-IDO end points for general Ids and subclasses with varying amounts of ZM-SZE

Zivv-Uerzieg mice obtained by outbreeding were divided into groups of 10 mice each. Each group received 30 μg of Ar 1-42-peptide at weeks 0 and 3. The first group received 25 μg of 529 ZE plus 10 μg of ZM-SZE; (o) the second group received 25 μg of 529 5E plus μg of CM-C5E; the third group received 25mcg of 529 ZE plus 0.Tmkg

OM-S5E; the fourth group received 25 mcg of 529 ZE plus 10 mcg of ZM-C5E in a priming dose and then 25 mcg of only 529

ZE in the second dose; the fifth group received 25 mcg 05-21; the sixth group received 25 mcg of 25-21 plus 10 mcg of ZM-S5BE. with

Mice were immunized subcutaneously in the buttock region with a total volume of 0.2 ml, divided equally into each of two areas at the base of the tail/buttock region. with

Mice were bled on days 0, 21, and 42. Reciprocal endpoint titers of anti-Ar1-42-peptide-IdDS for total "

Id9o and subclasses were measured in separate sera (p-10) at week 6. The results of the endpoints are shown in Table 9. The results of the Ids1 subclass are shown in Table 10. The results of the Ids2a subclass are shown in Table 11. (22) z y, h o z c 5 : 7 Before oo Eto o added tly ovo - and " First dose 529 ZE (25)45M-OSE (10); second dose only 529 ZE (25). se) g i ime) ! but -

F z o name) 60 vv 529 ZE (2534ОМ-С8Е (1) 116222 143140

" Before oo Eto o added tly ovo " First dose 529 ZE (25)-M-SOZE (10); the second dose is only 529 IU (25).

Example 6

Reciprocal titers of anti-Ar1-42-IdO endpoints for common dos and subclasses with 70 varying amounts of CM-SZE

Zm/izv-UUervieg mice obtained by outbreeding were divided into groups of 10 mice each. Each group received 300 μg of A1-42-peptide at weeks 0 and 3. During immunization at week 0, the first group received 500 μg 529

ZE; the second group received 5 omkg of 529 ZE plus 1 omkg of ZM-SZE; the third group received 5 µg 529 5E plus 5 µg

OM-S5E; the fourth group received 5 µg of 529 ZE plus 2 µg of ZM-C5E; the fifth group received 5 µg of 529 5E u5 plus 0.5 µg of M-SZE; the sixth group received 1905 5E. When immunized at week C, groups one to five received the same dose as when immunized at week 0, except that 529 ZE was reduced from 50 to 25 μg.

The amount of ZE received by the sixth group was increased from 195 at week 0 immunization to 1.295 at week 3 immunization. Mice were immunized subcutaneously in the buttock region with a total volume of 0.2 ml, divided equally into each of two sites at the base of the tail/buttock region. .

Mice were bled on days 2, 20, and 35. Reciprocal endpoint titers of anti-Apr1-42-peptide-IDO for total

IDO and subclasses were measured in separate sera (n-10) at week 5. The results up to the end points are shown in Table 12. The results of the IDS1 subclass are shown in Table 13. The results of the IDO2a subclass are shown in Table 14.

F

» sch h

F i - "sh Z s g" 4 -I

F v and m

Example 7

Immunization with TA-STI - and C4-MZ-peptide in rhesus macaques 60 The next experiment is planned to directly compare the number of combined forms of peptides and adjuvants in primates (rhesus macaques) to identify potential peptide/adjuvant combinations for the transition to clinical trials on a person Specifically, the adjuvant-containing form of 529 5E was evaluated against incomplete Freund's adjuvant (IRA) in combinations with (1) a peptide conjugate produced from 5IM-epm T-helper/Zim dad STI. (511-riis), which has the following sequence:

Zhyt im i chuk Bil Shi ide was Zh i ie y acer tia y v v Ilka (ШЧО ho Ж): or C4-MU3 peptide conjugate produced from HIV-1 (C4-MZdo br), which has the following sequence:

She Chil He saw Sha Yakr a biv twi ku lo Za Az Moi Yaut iv it lu ka sha dayu Ai

Chh Shu bl ish il ys id Bir Yage Shchizh zhu

Bil ty Yaukh vyv ke yshzh SK yiv zna).

Design of the study: 19 For this study, a total of 8 animals were used, 4 Mother-A"O1-- and 4 Mother-A"01-, as described in table 15. yuyu c 2 yuyu o yuyu

The animals of group 1 received 0.5 ml of Tp-STI - peptide (1.0 mg/ml) in a water-in-oil emulsion with 0.5 ml of IRA per ee, in a total volume of 1.0 ml. Animals of group 2 received 0.5 ml of 5T1-r11C (1.Omg/ml), combined with 240 μg of ZM-SZE human su, 5Omkg of 529 ZE with a final oil concentration of 195 in a total volume of 1.0 ml. Animals of group C received 0.5 ml of C4-MZdovr peptide (2.00 mg/ml) in a water-in-oil emulsion with 0.5 ml of IRA, final volume 1.0 ml. -

Finally, the animals of group 4 received 0.5 ml of C4-MZvoR peptide (2.0 mg/ml), combined with 250 μg of human ZM-SZE, Fu 5Omkg 529 ZE with a final oil concentration of 195 in a total volume of 1.Oml. 3o All animals were immunized by intramuscular injection with the schedule of administration at 0, 4 and 8 weeks. Peripheral blood samples were taken immediately before weeks 1 or 2 after each immunization to monitor induction

100 by tetramer staining, p11cS (Suz Tyg Rgo Tug Azr Pe Avp sip Mei; ZEO IYUO MO:3, amino acids 19-27), specific reactions of EGIBROT and STI - reactions of the total mass of the culture (Groups 1 and 2), as well as by reactions " peptide-specific antibodies (Groups 1-4). 50 Safety and tolerability of ZT1-P11SYAEA: Form 51T1-p11C-IEA, which was administered by intramuscular injection at the same site three times in group 1 animals, was associated with significant injection site reactivity. One animal (93x021) developed a 1.5 cm abscess at the injection site two weeks after the second immunization. Another animal (95x009) also developed a 2.0 cm abscess at the injection site two weeks after the third immunization, which broke through the skin and required dressing. 7 ЗТ1-р110-529 5Е/6М-С5Е: Form 511-р110-529 5БЕ/ЛЗМ-С5Е, which was administered by intramuscular injection in (se) one place three times in animals of group 2, was associated with minor adverse effects. Both group 2 animals had vomiting shortly after receiving the third immunization at week 8. No other adverse effects were observed. ka 20 Ca4a-MZvo br IEA: The C4-MZvo brIEA form, administered by intramuscular injection at the same site three times to animals of group 3, was associated with significant injection site reactivity. One animal (98p013) developed a 1.0 cm abscess at the injection site one week after the second immunization. Another animal (98p007) also developed a 1.5 cm abscess at the injection site one week after the second immunization. This abscess in the animal required drainage and dressing after four weeks.

SA-MZvo ver 7529 ZE/6M-SZE:

GF) Form C4-MZdobRn529 5E/ZM-SZE, administered by intramuscular injection at the same site three times to animals of group 4, was associated with only one minor adverse effect. One of the animals in group 4 o (95x011) had vomiting shortly after receiving the third immunization at week 8. No other adverse effects were observed. because in all animals of group 1 and group C, in which the animals received IRA as an adjuvant, significant reactivity of the injection sites was observed. These results indicate that 0.5 ml of IRA is poorly tolerated when administered by intramuscular injection at a single injection site. It should also be noted that 3 of 4 animals receiving 529 5BELZM-SZE as an adjuvant had vomiting at week 8 shortly after immunization. Although the anesthetic (ketamine) is known to be associated with emesis, no animal emesis was documented during the course of this study. At the same time, there is insufficient evidence to link vomiting in animals with any harmful effects associated with 529 5E/ZM-C5E.

Results: Induction of cellular immune responses (Groups 1 and 2)

Staining of p11C-tetramer of freshly isolated blood:

Before immunization and one and two weeks after immunization, peripheral blood was collected from all subjects

Mothers of A"01-positive animals (Groups 1 and 2) were screened for the presence of p'1C-specific SOZ'SOV8"

T-lymphocytes by staining the soluble tetramer of MHC Class I. As shown in Table 16, only one animal (93х021), among those that received ЗТ1-р11С-peptide in combination with IEEA, revealed the presence of immunization-induced cellular immune reactions in the unstimulated peripheral blood.

Table 16

Percentage staining of p11c-tetramer and p11c-specific reactions of EGISROT in freshly isolated peripheral blood

Week ABOUT Week 5 Week 6

Before immunization 1 week after the 2nd immunization 2 weeks after the 2nd immunization

Animal/group Form Coloring | EI IZROT | Dyeing of the tetramer EIZROT Dyeing | IZROT Number of 5ES per 106 tetramer Number of ZES (fresh blood Number of ZESTetramer (fresh blood cellsg | per 106 per 106 fresh blood cells cells 93x021 (1) ЗТ1-р11 CE 0.02 None 0.15 5b.3 012 21.9 data 98p002 (2) |8Т1-р11С529 0.05 0.01 6.3

ЗЕ/СМ-С5Е 98p002 (2) |8Т1-р11С529 0.05 0.04 15.0 0.01 9A

ЗЕ/СМ-СВЕ with 5 0000000000000007770000077000000777777777000700700070070700202020202020020о

Week 8 Week 9 Week 10 4 weeks after the 2nd immunization) 1 week after the 3rd immunization 2 weeks after the 3rd immunization

Animal Form Fresh blood EIZROT Fresh ESIZROT Fresh blood ESIZROT blood Ge) zo 9BHO0O (1) ZT1-r11 SE None 0.01 2.5 0.02 1.9 data se 93x021 (1) ZT1-R11SNEA 0.02 None 0. 14 No data 0.02 No data data « 98p002 (2) ЗТ1-р11С-529 No No data 3.8 He»)

ЗЕ/СМ-С5Е data 98t002 (2) 8тТ1-р11С529 0.02 None 0.02 |No data -

ZE/SM-S5E data

Shown in the form of a percentage of SOZ"СО87 T-lymphocytes of freshly isolated blood, which are stained positively with p11C-tetramer; MO, not performed. « p. - , i, nsch !

P11C-specific EHIBROT reactions: for an additional assessment of the induction of cellular immune reactions in animals of Group c 1 and 2, lymphocytes of freshly isolated peripheral blood were screened for the presence of p11C-specific :z" со3"с087 T-lymphocytes using the EI IZROT analysis. As shown in the table 16, only animal 93x021, which demonstrated detectable levels of pHO-specific CO8" T-lymphocytes according to the tetramer assay of freshly isolated blood, had detectable p11C-specific CO8" T-lymphocytes according to the EPIZROT assay. In each case, a positive reaction determined p11C-tetramer analysis, was confirmed by a positive p11C-specific reaction of EGI5ROT. ee, p11C-specific cellular immune reactions after stimulation with the p11C ip migo peptide: In an attempt to increase the number of p11C-specific cells before the analysis, freshly isolated peripheral blood lymphocytes were stimulated ip 5or Mygo with the p11cS peptide and gp--2. After 14 days, the obtained effector cells were screened for binding to p11C-tetramer, and also for p11C-specific lytic activity in a standard STI release assay

F of chrome. Results of p11C-tetramer analysis and STI functional analyses. shown in Table 17. Animal 93x021, which consistently showed p11C-specific immune responses in freshly isolated lymphocytes, demonstrated a very high level of tetramer binding and functional STI activity one week after the second immunization. This indicated the induction of a highly effective p11C-specific cellular immune response. In contrast to the results observed in freshly isolated lymphocytes, one animal from group 2 (98p008), 5T11-p11C0-529 (F, ZE/ZM-C5E), began to show evidence of p111C-specific cellular immune responses two weeks after the second co-immunization. However, the p11C-specific immune response observed in the group 2 animal was significantly lower than that observed in this responding group 1 animal. 60

Table 17

Percentage staining of p11cS-tetramer and functional p11C-specific STI reactions after stimulation with p11cS ip migo peptide

Week 0 Week 5 Week b

Before immunization 1 week after 2nd immunization 2 weeks after 2nd immunization bo Animal/group Form Staining STI 20:11 Esti Staining sti 201 Staining | STI 201 tetramer Z tetramer ET tetramer ET ee re re ru rr rr data to be used) ovTlRysEA 005000000000z000663 vyv 0265 98p002 (2) ЗТ1-р11С-529 0.21 no data no 0.73 no

HIN i NI NNYA PON nan mV PON ee shi b sho o

ЖЕ/СМ-С5Е data data data nnnshnSzhzhyaayaYUSHEshdLllVIIOOOOOOLLLLVLVVVVVVVV 05 Cannes Kentsnia Kdnnnnia 706 4 weeks after the 2nd immunization 1 week after the 3rd immunization 2 weeks after the 3rd immunization of tetramer ET ET tetramer ET and ibn autumn YOU NNNONNNY SHOBY NIKI PV data : data he sent the data to CHANGE NBN SONGS NO

ЗЕ/СМ-С5Е of your data with an TIN NBN NBN NO

ZE/SM-C5E of the data and Pettnyutsityu tt tyatin are shown in the form of the percentage of СО3"7СОВ8 7 cultured cells that are stained positively with p11С-tetramer.

Analysis of intracellular cytokines: for additional characterization of the functional and phenotypic properties of immunogen-induced peptide p1 and 1C-specific lymphocytes, the intracellular expression was subjected to monitoring for the expression of Tpi-type cytokines IRM-y, TMEo, 1-2 and TN2-type cytokine 1-4.

Intracellular expression of cytokines was monitored in peripheral blood lymphocytes after initial stimulation of ip migo in the presence of TOμm peptides p117C and gpII/-2. Then these cultures were maintained for 14 days with 40 U/ml II -2. After two weeks, the cultured cells were stimulated either with medium alone or with 30 1 µM peptide p11Szhapi-Nitap CO28 and apii-pitap CO494 for one hour. One hour later, these cells were treated with Brefeldin A for an additional five hours to allow intracellular cytokines to concentrate in the endoplasmic reticulum. Intracellular expression of cytokines was then quantified by flow cytometry (Tables 18 and 19).

As shown in table 18, two weeks after stimulation with the peptide ri1C ip mygo POP" lymphocytes

From the peripheral blood of animals of group 1 93x021 (5T1-p11C-RA) showed a low level of expression of TN1-type cytokine, and less than 1.595 of all cells actively secreted IEM-u, TMEo, or II-2. Approximately 895 of all POP" lymphocytes after stimulation with p11C peptide ip could actively secrete cytokine Tp2-type 1-4, and it was found that the secreting II-4 cells were restricted to the subpopulation of POP"CO4" lymphocytes. After short-term re-exposure to the p11C peptide, subpopulations of ri1!C-tetramer "7 and СО3'СО8' lymphocytes actively secreted Tp1-type cytokines of IEM in Her TMEo, but could not be induced to secrete significantly increased levels of 1/-2. c After repeated exposure to the p11cC peptide, the secretion of Tp2-type II-4 cytokine did not change. ;" 7 zpotetnerye 0090 ovo 0 0oovo o vsayalenikhova 00 0ovvet velo va 2 yu i

Ф nnnnnnnnnnnnnnshShnjzhyayashyaShyayaoyanShnnnyayayaaaaaaaaaaa with o name) in os: INNY NI NN NI NNYA NIKI NO I NO NN NS z єso3so4 3310 3789 11 1428 2567 1.8

25.1. Stimulation index.

b Shown as the number of the indicated cells that stain positively for the indicated cytokine, per 109 SOZ34 cells, minus the background staining (isotypic control).

As shown in Table 19, two weeks after stimulation with the peptide ri1C ip myo, SOZ "7 peripheral blood lymphocytes from animal 98p008 group 2 (5T1-p11C0-529 5BE/LZM-SZE) showed a low level of expression of TAI1-type cytokine, and less 1.095 of all cells actively secreted IEM-u, TMEo or 1-2. Interestingly, approximately 2095 of all ХОЗ" lymphocytes actively secreted Tp2-type cytokine 1-4 with a 2.5-fold increase in the number of ІІ/-4 producing cells compared to animal of group 1. As in the case of the animal of group 1, it was found that the secreting 1/-4 cells are limited to the subpopulation of SOZ 7CO04" lymphocytes. After short-term repeated exposure to the p11C peptide, the subpopulation of ri1C-tetramer" and 203708" lymphocytes of the animal from group 2 could stimulate the secretion of TME0, but not IRM. In contrast to the animal of group 1, after repeated exposure to the peptide, a significant increase in the expression of I-2 C0O3"C08" by lymphocytes could be observed. As in the case of the animal of group 1, after repeated exposure to the p11cC peptide cytokine secretion inu Tp1-type II--4 did not change. p 29 Results: Immunogen-induced humoral immune reactions (Groups 1 and 2): Ge)

To evaluate the induction of immunogen-specific humoral (in the form of antibodies) reactions, the titers of EGI5ZA anti-ZT1-p11C antibodies were measured in the serum of animals of groups 1 and 2 immediately before immunization and 1 and 2 weeks after the second and third immunization. The results are summarized in Table 20

Table 20 p

End point titers of EGISA serum from rhesus macaques immunized with ZT1-r11s (groups 1 and 2) with Ш0 ЛШОШОШО2О2 1 ЛО ЛО ОИ- 0 " "О rо lo lo bloo t nyn t togo " - is ШТ « ШЧ" 2 2 lll ju 20 5120

И» 111111 p I PO NOTI tvoo a 001020 98pО08 2 8Т1-р11с2529 5Е/СМ-С8Е 5 200 -i nn nn a PO p PO

Ф ШТ " " (" 2 l5 OZ tvo " ny nyshshshiyini ni ly blo vo 70 a Endpoint binding titers of antibodies in the form of values reciprocal to the highest dilution of plasma that gives a reading of 00 experiment/control (E/C)"3 ,0.4)

Immunogen-induced humoral immune reactions (Groups C and 4):

To assess the induction of immunogen-specific and adjuvant-specific humoral (in the form of antibodies) reactions, 29 EPIZA titers of anti-C4-UZvo vr and anti-cM-SZgR antibodies were measured in the serum of animals of groups C and 4 directly

HF) before immunization and 1 and 2 weeks after the second and third immunization. The results are summarized in Table 21. The results show that the maximum titers of Ca4-MZvo.vr-antibodies in plasma were generated in the first week after the second immunization in all tested animals. The maximum titers of antibodies in plasma in animals of group C (C4-MZvo.vr'RA) were several orders of magnitude higher than the maximum titers of antibodies in plasma observed in group 4 (C4-MZvoevr 529 ZE/ZM-C5E) . Group 4 animals showed low but detectable levels of anti-CM-C5R antibody titer, peaking one week after the third immunization.

Table 21

End point titers of EGISA plasma from rhesus macaques immunized with C4-UZvo.vr (groups C and 4) animal/group Form Week Titer of C4-UZ antibodies Anti-pitap-cM-SZE-AB titer in I PO I MON No sleep

RO PI POLON CANVAS POLO NNYA PON sv 09111156 yuyu 11111116

I

1001 01111115 p 1nvyu011111yu vyyu vvy yuyu |00 experiment/control (E/S)»3.0.

The induction of neutralizing antibodies in animals of groups C and 4 was also monitored; the results are summarized in table 22. These results show that both animals of group C and animals of group 4 developed neutralizing antibodies that were capable of neutralizing the ZNIMdo virus in mice. The titers of ZNIMvo-neutralizing C antibodies observed in animals of group 3 were generally higher than those observed in animals of group 4. In addition, after the final immunization, the serum of three animals from group C showed a low level of neutralizing activity against ZNIMvo v-strain of this a virus that is difficult to neutralize.

Te z 10111 neiraliyuchanitlyayu 00000000 -

EE IN SSZ PN YA Oslo NO MAN 00150008 el PO PO NO NN NO NO sti KI 11 tt

ESSAY MEANS MONDAY MONDAY NOVEMBER FRIDAY: in 1st 1

PO MON PO: MON MON PO - 111 1v i» Jan I EP ZY MON

ES SE ste ZNNNNNY NONOSLONNNNYA PON PO

RN PIPPNNYA POLON KON ONNN KON s NO set - PO PON PO: PON PO NN PO 1111111 that PI PON NOT ON NOT PO ote 000000 bbyuvvomoy 00000000 "et IN POM PON CO TONN NO CT NO tp 611111 shi PO PON POT POT PO yuyu 1 . o measured by the absorption of a neutral red dye. ka Example 8

Study of therapeutic efficacy on ROAPP transgenic mice treated with Ар1-42 and adjuvants in The following experiment was designed to compare the number of combined forms of adjuvants in ROAPP transgenic mice to test the therapeutic efficacy of Ар1-42-peptide.

Design of the experiment: ROARR transgenic mice aged 10.5-12.5 months (male and female) were divided into four groups of 40 mice, sorted in such a way that each group matched each other as closely as possible in terms of age, sex and transgenic parent . These groups are described in table 23: b5

Table 23

Ар1-42-peptide was obtained from EIap Rpagtaseciis!v, 529 ZE and MRI m ZE were from Sogih and mouse COM-C5ZE 70 was from Viozotsgse. All mice received injections at weeks 0, 2, 4, 8, 12, 16, 20, and 24. Mice were bled 5-7 days after injection, beginning after the second injection. Groups 1, 2 and C were injected subcutaneously with a volume of 200 μl, while group 4 received doses of 250 μl subcutaneously. Animals were killed on week 25 of this study. Titers were obtained using a dilution giving a maximum reading of 5095 optical density.

Results:

Immunogenicity and response in the form of antibodies: All groups reached their maximum geometric mean titer (GMT) of antibodies to the A1-42-peptide either at the second (KS529-OM-C5E) or at the third blood removal (MR tm BE, MRI tm BENOM-C5E ) (see Figure 1). At the maximum OMT, MRI tm ZENOM-S5E was 16400, then 529

ZENOM-S5E was 13,400 or about 1.5 times the MR control! tm 5E 9700. However, these titers on two OM-C5P-containing forms (groups 2 and 3) fell to approximate levels of MRI tm ZE-control with equine SMT MRI tm

ZE-4600, MRI tm BZENOM-S5E-5350, 529 BENOM-SBE-4650.

To determine whether the final decrease in the titer of the two ZM-C5E-containing forms was due to the reaction of anti-SM-C5BR antibodies, EGISA was used to determine whether anti-ZM-C5B antibodies were formed during immunization. Sera from all animals receiving SM -C5E, titrated against murine COM-C5E, C 259 used throughout this experiment. No evidence of anti-ZM-SZE antibodies was obtained in any of the treated animals (data not shown).

AR Levels in the Brain: All three treatment groups significantly reduced accumulation as a whole

Ar-peptide (Figure 2 - individual results; Table 24 - combined results) in the cortical region of the brain of the ROAPP mouse. Assays of EGISA AR (total and form 1-42) were performed as previously described (54) with ee, using guanidine-solubilized brain homogenates. Statistical comparisons were performed with Ha using the Mann-Whitney probability test. "

F with vve | meive Mmrivegomove vegohmosove in decrease 12011861 «

The value of P (M-M/) 37 0.000135| . 02006808 from VeluiaR (MUYU 00 oo svo sob mo oo 4) Amyloid load: The degree of amyloidosis was determined quantitatively in the frontal part of the cerebral cortex using immunohistochemical methods, as previously described |55). All three treatment groups found a significant reduction in amyloid burden (Figure 4 - individual results; Table 26 - pooled results). o yuyu 11111000 ve

Medianasyavu | 7980 ble 000obo 00 in decrease | in00o1ve5

VelichnaR(MUYU 00000010 00001 65 Neuritic load: The effect of processing on the development of neuritic dystrophy in the frontal part of the cerebral cortex was evaluated immunohistochemically, as previously described |55). All three treatment groups significantly reduced neurite burden (Figure 5 - individual results; Table 27 - combined results). ; 0000001oeve |MRIve Mi EhoM.See 529 ZEhOMSVE, decrease | 20066885 about VeliinaR (WASH 000s0015a with myself 00sooot

Astrocytosis: The degree of astrocytosis in the retrosplenial part of the cerebral cortex was quantified as previously described |55). Treatment groups containing ZM-C5E showed significantly reduced astrocytosis (Figure 6). 75 Example 9

Immunization with peptide CA(EZU)-MZvz/vr of macaques

The purpose of this experiment was to evaluate the immunogenicity of the peptide conjugate produced from Nim-1 Epm

CA(E9M)-MZvz br, which is administered with the combined form of adjuvants of this invention to another species of primates, dog-headed macaques (Masasa Havsisciagiv). C4-MZvz br-peptide, described in example 7, was modified by replacing glutamic acid in amino acid residue 9 with valine. The obtained peptide conjugate named CA(E9M)-

MZvz vo, used, and it has the following sequence: shcha id kiv Zh Mn Sho pr bin chay az schu sch

Shchy Bi Miy Tut Al che ie t z. by. for about

She Me She vdlyu she GP Yar iho ku aku

From those girls Zhe (VID l so) I . with, and and I, and and and

Replacement of glutamic acid with valine (E9M) in the C4 region of this peptide increases the immunogenicity of the peptide above the CM of the immunogenicity of the unmodified peptide in the mouse model. -

This Epm peptide produced from NIM-1 is capable of inducing humoral immune responses in mice. However, due to the difficulty of extrapolating the results obtained in mice to humans, it is necessary to test the potential (22)

HIV-1 immunogenic compositions in primates (non-human) before transition to human Phase I clinical trials. In this experiment, both intramuscular (IM) and intranasal (IM) routes of administration were evaluated. Animals were immunized five times on weeks 0, 4, 8, 18 and 23. Once a week up to week 25 inclusive, blood samples and cervical-vaginal and mucosal washings were taken and analyzed for the presence of antibodies to this immunogenic composition.

Design of the experiment: A total of eight cynomolgus macaques were used for this experiment. Group 1 "70 received adjuvant; group 2 received the form of adjuvant 529 BE plus OM-S5R. Animals were housed and evaluated in animal care facilities. with" with? Immunizations: All intranasal immunizations were delivered using a nasal spray device with measured doses of 100 μl. All intramuscular injections were made into the quadriceps muscle using a needle and syringe. All animals were immunized according to the scheme 0, 4, 8, 18 and 23 weeks.

F Forms:

Group 1: 1000 μg of CA(E9M)-MZvo vr-peptide in sterile normal saline solution, final volume 200 μl (100 μl in each nostril).

Group 2: 1000 μg C4(EZ9M)-MZ in vr-peptide, 50 μg 529 ZE and 250 μg human ZM-SZE, final oil concentration 195, final volume 1.0 ml (500 μl into each quadriceps muscle by IM injection) . (F) Assays for monitoring immunogen-induced immune reactions: Immediately before and after immunization, all d) animals were carefully observed for immunogen-induced humoral immune reactions using the following assays: (1) Determination of serum titers of anti C4(E9M)-MZvo vr-peptide-Ido-antibody using EGISA. (2) Determination of mucosal (cervical-vaginal, nasal) titers of anti-CA(E9M)-UZ vo'br-peptide-Ids-antibodies with the help of EIGHIZA.

Results: Safety and tolerability of the immunogen:

CA(E9M)-MZvo vrpeptide when administered alone or in combination with 529 ZE/ZM-SZE adjuvants was very well tolerated. In animals immunized intramuscularly using a needle and syringe, adverse reactions at the injection site were not noted. All animals were closely monitored for changes in body temperature for 24 hours immediately after each administration of the immunogen. At no point in time during the study did any of the animals show abnormally elevated body temperature readings.

Immunogen-specific reactions in the form of serum antibodies:

Serum samples were obtained from all animals immediately before and one and two weeks after each immunization (within 25 weeks). Two weeks after the final immunization, all serum samples were analyzed for the presence of anti-C4A(E9M)-MZ-peptide-IdsC-antibodies. Intranasally immunized animals of group 1 (only

C4(E9M)-MZvo vr-peptide) did not show higher titers of anti-C4(E9M)-MZvo vr-peptide-IdC-antibodies..- in serum than non-immune levels (Figure 7). In contrast, animals of group 2 (IM-introduction of 70. CA(EUM)-MZvo vr-peptidun529 ZE/ZM-SZE) developed significant levels of SEZM)-UZ specific Ids-antibodies, (Figure 7).

The geometric mean titer of the reported endpoint was calculated using the lowest titer from each individual animal that is 3-fold greater than that of the pooled "naïve" serum at the same dilution.

Group 1 animals (without adjuvant) had titers of aHga-SA(E9M)-MZvo.br-Idb-antiTtt in cervical-vaginal lavage samples that were higher than non-immune levels only after the fourth immunization, but decreased thereafter (figure 8). In contrast, group 2 (adjuvanted) animals had anti-CA(EZ9U)-UZvovr-IdO antibody titers in cervical-vaginal lavage samples that were higher than non-immune levels after the first immunization and increased after each subsequent immunization (although there was some later decline in each case) (Figure 8).

Animals of group 1 (without adjuvant) did not show titers of anti-C4(E9M)MZvo vr-I9C antibodies in nasal washes higher than non-immune levels (Figure 9). In contrast, group 2 (adjuvanted) animals developed significant levels of anti-C4(E9M)-UZ 89 vr-Ids antibody titers in nasal lavage samples (Figure 9).

Bibliography 1. Moztapp, T.K., ei ai., 9. Ittipoi., 136, 2348-2357 (1986). sch 2. 0.5. Rayepi Mo5057540 3. 00.5. Rabepi Mob113918. i) 4. Apiegv, 9.O., ei ai., U. Ittipoi., 158, 3947-3958 (1997). 5. Zspagiop-Kegaep, T., ei ai., U. Ittipoi. 154, 5320-5330 (1995). 6. Spaiyiv, NMU., ei ai., U. Ittipoi., 154, 4623-4629 (1995). so zo 7. Mitau, NMU, apa Nagirgazpaa, .., U. Ehr. Mea., 181, 387-391 (1995). 8. 0.5. Rabepi Mo5571515. with 9. 0.5. Rabepi Mo5723127. "E 10. 0.5. Rayepi Mo5976539. 11. RipkeItap, R.O., apa Noitez, 9., App. Keum. Ittipoi., 8, 303-333 (1990). Me) 12. Zparreg, SM., ei a!., U. Ehr. Mea., 175, 1367-1371 (1992). 13. Korauazvnpi, M., ei a!., U. Ehr. Mea., 170, 827-845 (1989). 14. Rybiizpea Ipiegpaiopai! Rybepi Arriisayop MoUUO 90/05147. 15. O.5. Rayepi Mo5078996. 16. 0.5. Rayepi Mo5229496. « 17. 0.5. Rayepi Mo5073627. from p. 18. AIdegzop, M.K., ei a!., U. Ehr. Mea., 178, 669-674 (1993). . 19. Zparreg, SM,, ei ai., U. Ittipoi., 154, 5842-5850 (1995). "» 20. O.5. Rayepi Mo5679356. 21. Rybiiznea Ipiegpaiopai! Raiepi Arriisayop Mo M/O 00/69456. 22. 0.5. Rayepi Mo5013548. -I 23. Sh.5. Rayepi Mo5019387. 24. Spaghii, A. . doppzop, K.R., ei ai., 9. Migoi., 68, 3145-3153 (1994). 27. Esheg, O.N., ei ai., AIOZ5 Kez. Nit. Keigomigizevz, 10, 1433-1441 (1994). ko 28. Veggoi»vKu, U.A., ei ai., U. Siip. Ipmevi, 88, 876-884 (1991).

F 29. RaikKeg, T.U., ei ai., U. Ittipoi., 142, 3612-3619 (1989). 30. Nagi, M.K., ei a|., U. Ittipoi., 145, 2677-2685 (1990).

C1. Naupez, V.R., ei ai., 9. Ittipoi., 151, 1646-1653 (1993). 5B 32. Naii, M.K., eig ai!., Rgos. Mai). Asad Zsi., OBA, 88, 9448-9452 (1991). 33. Vapshey, .A., ei ai!., AIO5, 12, 1291-1300 (1998). (F. 34. Naupez, V.R., eg ai., AIO5 Kez. Nyt. Keigomigizev, 11, 211-221 (1998). ko 35. 0.5. Rayepi Mo5861243. 36. 0.5. Rayepi Mo5932218. 60 37. 0.5. Rayepi Mo5939074. 38. 0.5. Rayepi Mo5993819. 39. 0.5. Rayepi Mob0O37135. 40. Ryriiznei Eigoreap Raiepi Arriisayop Mob71947. 41. O.5. Rayepi Mob024965. 65 42. MiSheg, M.O., ei! ., U. Ittipoi., 147, 320-329 (1991). 43. Kygoda, M..)., ei a!., U. Ehr. Mea., 187, 1373-1381 (1998).

44. Pao, N-H, ei ai., 9. Migoi., 74, 254-263 (2000). 45. Biaayiv, N.R., ей ай., 9). Ittipoi., 157, 462-472 (1996). 46. Rogdadog, A., ei ai., U.

IttypoY., 158, 834-841 (1997). 47. Rybiizpea Ipiegpaiopai! Raiepi Arriisayop MouUUO 9/27944. 48. O.5. Rayepi Mo4666829. 49. Satevs, O., et al., Maishige, 373, 523-527 (1995). 50. Rybiizpeid Ipiegpaiopai! Rayepi Arriisayop MoUUO 98/20734. 51. 0.5. Rayepi Mo5830877. 70 52. Rybiizpey Ipiegpaiopai! Rayepi Arriisayop MoUUO 99/51259. 53. Sh.5. Rayepi Mo5593972. 54. doppzop-Mosa), O., ei ai., Rgos.

Mai! Asad

Zsi., OBA, 94, 1550-1555 (1997). 55. Zspepk, O., et al., Mayige, 400, 173-177 (1999).

Claims (67)

19 Formula of the invention 1. An antigenic composition that contains a selected antigen from a pathogenic virus, bacterium, fungus, or parasite, or from a cancer cell, or a tumor cell, or from an allergen, or from an autologous molecule and an effective adjuvant (immunostimulating) amount of the combination of (1) aminoalkylleucosamine phosphate compound (ACP) or its derivative or analog and (2) a cytokine or lymphokine or an agonist of said cytokine or lymphokine, where this combination of adjuvants enhances the immune response in the vertebrate host to said antigen. 2. Antigenic composition according to claim 1, where the selected antigen is a polypeptide, peptide or fragment produced from a protein. high school 3. Antigenic composition according to claim 1, where AOR is used in the form of a stable "oil-in-water" emulsion. 4. Antigenic composition according to claim 1, where the cytokine or lymphokine is selected from the group consisting of (o) granulocyte-macrophage colony-stimulating factor and interleukin-12. 5. Antigenic composition according to op. 4, where oxytokine or lymphokine is a granulocyte-macrophage colony-stimulating factor. so so 6. Antigenic composition according to claim 5, where AOR is used in the form of a stable "oil-in-water" emulsion. 7. Antigenic composition according to claim 4, where the cytokine or lymphokine is interleukin-12. with 8. Antigenic composition according to claim 7, where AOR is used in the form of a stable "oil-in-water" emulsion. " 9. Antigenic composition according to claim 1, which additionally contains a diluent or carrier. 10. Antigenic composition according to claim 9, where AOR is used in the form of a stable "oil-in-water" emulsion. b" 11. Antigenic composition according to claim 1, where ASR is 2-KK)-Z-tetradecanoyloxytetradecanoylamino|ethyl-2-deoxy-4-0-phosphono-3-0-(K)-Z-tetradecanoyloxytetradecanoyl|-2-KK )-Z-tetradecanoyloxytetradecanoylamino|- E -Ob-glucopyranoside (529). 12. Antigen composition according to claim 1, where the selected antigen is an antigen from the human immunodeficiency virus (HIV). " 13. The antigenic composition according to claim 12, where the selected HIV antigen is an HIV protein, polypeptide, peptide or Z fragment obtained from the specified protein. with 14. The antigenic composition according to claim 13, where the selected antigens are HIV peptides selected from the group consisting of peptides having the following amino acid sequence: Suya Tyh Aku RKO Aa Tuh aets Tsuv Ago tui Aku yI16 t niya 116 41u tgo Ch41u Aku kia Bi Tuh Tvg Tv uk (880 Her? і» MpOo: 1): m) Ishe Ba T16 І124 Ana Shab thr biv biu Uai biu Tscuye z1a meh F Tuk Aia Tit Ak ko Ana tTuh Anv tsuzhv Ah Mtzh Ak Iza niv I1ia 01u iho 0b1Uu Aku Aid Ba Tuk Tih Tk Il (880 Ii» 5» Shol1)2 o AK 9413 T16 x10 Ana Jok Tkr Mia ShPuv uai o1u Gu lyad chai t Tuk va ich 91u Suye Zhyh go Tut ayur 7116 Ani siv Me yi4u go (ShZHO hv MOkh31, b5 i: Iiee SBU Rjo Ch1u Ah Aa ybe Tut Via Ahu Aty (850 IV SHOS4)z and uv bij T1e her But Maye Tkr bip tuvi chni b1u muye viv Maye yu Zhuk Aza tii Avu ho Asht Avo Avp izh A Si Agu Imlpi Bog 116 01u ?ko 01U Aga Aia ba Tukh ata Atu ykYaa (SHO hv MO: 5. . 15. Antigenic composition according to claim 14, where the HIV peptide is a peptide having the amino acid sequence: uv 415 116 x1i6 Av Me gr bip 41z uv y1u tsuye Aza mob tut Aia Suye bik Asu Rho Aly Tukh Aip Shu Aga tsu Aku tiv ho Liv 16 91u rho 41u ku Aia Te Tukh Tit Tyu Tsuv (HO i0 that 1 g 16. Antigenic composition according to claim 14, where the HIV peptide is a peptide having the amino acid sequence: 63zu ho 215 ABU liv Viv Tukh Zhnh Chnh uzih (8EO Ir MOo:2). heh 17. Antigenic composition according to claim 14, where the HIV peptide is a peptide having the amino acid sequence: sch ka Fi» I1F x16 Av Tykh Tyr niv Tsuv tni 013 I vp chai ch Tuh tes Phys 41u Suv Tk Zhgo Tuh Avr I1F9 ALyayu 012 Meb yivts F from the VEO TR of the MoS). m 18. Antigenic composition according to claim 14, where the HIV peptide is a peptide having the amino acid sequence: eat bid 519 116 vev Sha: tThr ip im chai b1u Iuv vza mah « Tut Viv ЖЖ Dhb yko Ma laz vp Tyh Ak bim lho Ba bah - o chav schu M b1U is aty miv ta tuh Aza dgv ar (WHAT they "from ip4,. " 19. The antigenic composition according to claim 14, where the HIV peptide is a peptide having the amino acid sequence: with. Ye Fiz I1F IZ Av Me Thr biv tuai tUAi bizh iuya Ata Mah V tuh Aza TiK Ahd Rho Or Ave Aa Tk Avu 014 aku wa zhh I«e) . k t1ia 91U Rko schu aku Kish Zh» Tukh AiTa Aho Aku (EKO IB 2 SHMO:8). at 20. Antigenic composition according to claim 12, where ACP is used in the form of a stable "oil-in-water" emulsion. F 21. Antigenic composition according to claim 12, where the cytokine or lymphokine is selected from the group consisting of granulocyte-macrophage colony-stimulating factor and interleukin-12. 22. Antigenic composition according to claim 21, where the cytokine or lymphokine is a granulocyte-macrophagic vv Colony-stimulating factor. 23. Antigenic composition according to claim 22, where AOR is used in the form of a stable "oil-in-water" emulsion. F) 24. Antigenic composition according to claim 21, where the cytokine or lymphokine is interleukin-12. ka 25. Antigenic composition according to claim 24, where ASOR is used in the form of a stable "oil-in-water" emulsion. 26. Antigenic composition according to claim 12, which additionally contains a diluent or carrier. 60 27. Antigenic composition according to claim 26, where AOR is used in the form of a stable "oil-in-water" emulsion. 28. Antigenic composition according to claim 12, where the AOR is 529. 29. The method of increasing the ability of an antigenic composition, which contains a selected antigen from a pathogenic virus, bacterium, fungus or parasite, to induce an immune reaction of a vertebrate host, which involves the administration of the antigenic composition according to claim 1 to the indicated host. 65 30. A method of increasing the ability of an antigenic composition, which contains a selected antigen from a pathogenic virus, bacterium, fungus or parasite, to induce an immune reaction of a vertebrate host, which involves the administration of the antigenic composition according to claim 9 to the indicated host. 31. a method of increasing the ability of an antigenic composition containing an HIV antigen to induce an immune response of a vertebrate host, which involves the administration of the antigenic composition according to claim 12 to the specified host. 32. A method of increasing the ability of an antigenic composition containing an HIV antigen to induce an immune response of a vertebrate host, which involves the administration of the antigenic composition according to claim 26 to the specified host. 33. The method according to claim 32, where the selected antigens are HIV peptides selected from the group consisting of peptides having the amino acid sequence: o a ba g1e T16 Aep Meb bhr 013 a15 Uai F1u Tsuv yiv maei Shiv 119 TU Rko 41U Agu Aa UBo tTukh Tit TE tsuv (850 Tr y MO11), Ia biv tiv G1F Avo Mab Tgr bid 015 uaz su yuye Aza has tukh A14 TzhZ Ak KO Ani Tukh Abyu uv Atu Tsuv yku i16 Shchiv m a Ya1U ho FIUu Asu Ala Rio ZUuh Teh Those already (00 Iv shzoz2)1. Ahu ba 16 He liu Tk Ter Mia uv tai O1U 18 dep chai » LIZH yech 913 91U Su TIE Rgo Tut Mer I16 aka biv Meb tt S (Java IYU MO:3)" o Mu" 415 110 T16 Ayez Meb Tur biv biz Uvi s1u tsua zala mob Tut Aiva Tk Ahd RKO Ve Ava Avp Tzh Vu 010 Agu her tv o » Zhe 61U Ugo 01U Agu aa sch Zhe Tut A1a Aku lk (MO ho MO:4),; shcha z » Zhuzh Via Tvg Ahd rko Av vep Av obvk aku Fiv hu tlia vek TiF Zh1u ?ho 01U Agb Aa Br Tut Aza Aku Ak (ЖBO I MO). " " 34. The method according to claim 33, where the HIV peptide is a peptide having the amino acid sequence: -o s uv Chi x1e Y16 Avo Meb Tkr Oz Fiz wai O1u Shshshuzv liv moe xz Tuk ia Suye Tizh Aku Rko Av Tukh Anyu Shut Agu uv Ago I18 y niv і1z 0і1u Rgo Fiu au At ?ia Tuk Tx Ttg Muv (BEO Ii? - MI. s 35. The method according to claim 33, where the HIV peptide is a peptide having the amino acid sequence: v Tsuye ip 16 116 lap She Tlo biv bi Uki b1u ila Aza Maye mu Tuh Kia Tik MKU RKO Ana Tuh Avp Kui Ag Tuz Ako Iza miv Fu yi 52uU Rko b1Z Ahao Aza yo Tuk Tvhu Te yiuv (BO I» MkO:2,. rya 36. The method according to claim 33, where the HIV peptide is a peptide having the amino acid sequence: o Ak Fiv t1ie T1i4 sha ty yisr miv uv Ugi 01u uv lka chai yu tuh iz Phi 41U Sue Tit Rho Tuh Aer Ii Lip Pip Me: im ( vEo g MO). 6o0 37. The method according to claim 33, where the HIV peptide is a peptide having the amino acid sequence: b5 Tsup Yama yi1a I16 Avo Mob tTkro a1v via tuai S3u im left Mob " lived Yaku ho yiu ko Aza ia Tukh Aza Ah yhu (VO iz that 4, 38. The method according to claim 33, where the HIV peptide is a peptide having the amino acid sequence: o B biv 119 T1yu Aet Me Thr bip chai az C1u Tsuye Aza meh Tukh Aa t: Aku Rko dip Ato Ana Tk Agu Sa Aka Ia Yachh ia 41u iho hu Atyy y1ia y Tukh Ala Ah aku (AS YIV MO13). 39. The method of increasing the ability of an antigenic composition, which contains a selected antigen from a pathogenic virus, bacterium, fungus or parasite, to induce cytotoxic T-lymphocytes in a vertebrate host, which involves the administration of the antigenic composition according to claim 1 to the indicated host. 40. The method of increasing the ability of an antigenic composition, which contains a selected antigen from a pathogenic virus, bacterium, fungus or parasite, to induce cytotoxic T-lymphocytes in a vertebrate host, which involves the administration of the antigenic composition according to claim 9 to the indicated host. 41. A method of increasing the ability of an antigenic composition containing an HIV antigen to induce cytotoxic T-lymphocytes in a vertebrate host, which involves administration of the antigenic composition according to claim 12 to the specified host. 42. A method of increasing the ability of an antigenic composition containing an HIV antigen to induce cytotoxic T-lymphocytes in a vertebrate host, which involves administration of the antigenic composition according to claim 26 to said host. 43. The method according to claim 42, where the selected antigens are HIV peptides selected from the group consisting of peptides having the amino acid sequence: kha yil tiv tiv vi Mob Tr bin sich Uai b1u tsuv Aa Ma o zo Uk Aza Su Tit vka teo Ma cap MECH Tsuv Ah H1F o s Shiv 15 61U Rgo b1iu dhb ATA Rbe Tuk tkh TBh uv (950 19 shos1); F zv I1e giv Aa Mes Tkr bil obi uai 91u uv Aza mob ge Tukh A1a Tkh Ak Rho ABa Tuk Ayep Tsuye Ahu Tsuv Agu I16 niv I15 F1Uu Rho F1uU Ak Aza na Tuk Tikh Tikh Kul (VM) 10 « Mog2), - " 7 aku iv Hm s Zhe Mal Tyh Thr niv Tsuh chai 91u Tsuye Av chai (shko ho scho:x3), - aa Fiv yiI1v H1e Ali She Ttr 915 91 chUai O1u goes Ata has se Tuk viv Tkh AtO RKO Aea Avp Ato Tak AGO Sich koila Veh 0 WHAT) o; 7 42) Ka biy T1o y1F Avy Mei Thr Fiv Chai Chai b1u uv Aza Mob uh Aia ih ba. y Ved go Mp, Avy Ma Ti aku "41 Ahu ili cekh o I1F 01u yho O1u Ak9 dia Zhe Tukh Kia Ahu Ak (WHAT Ir yuyu claim 5,. 44. The method according to claim 43, where the HIV peptide is a peptide having the amino acid sequence: o B Phys T1i6 Ti Ava Me; hr biv 015 uaz slu Shu Aza mah viz song, Ansh Ro Ma Tukh Avo ShTsue Ahu Tsuv Hu 16 45. The method according to claim 43, where the HIV peptide is a peptide having the amino acid sequence: Ma Tkr bip 41z Uvi Ya1u Buv lia Meb led also Ahsh RKO Liu Tut Lev Kui Aku Ttsuv aku yi niv - 118 01U Rho u hu Kia Re Tukh Tk Tk tsuye (62O I Mo:2). 46. The method according to claim 43, where the HIV peptide is a peptide having the amino acid sequence: ki bi X1i6 i16 zva to Tih trnia Tsui chai 41u tsuye Avp tuvai Tut food Fiz 01u Suye Tak Rgo Tuh Ar I19 Av bii Mah food (ka go Жос3),. 47. The method according to claim 43, where the HIV peptide is a peptide having the amino acid sequence: schuye ip 16 I16 Aqua Mob Tkr biz bi ai F1u iua liv at 720 pog4). " 48. The method according to claim 43, where the HIV peptide is a peptide having the amino acid sequence: 16 gu Rho S1u Ahu viya Mao tuk Aza Aho ahu spo tv Mo: 5). 49. The method of increasing the ability of an antigenic composition containing a cancer antigen or a tumor-associated antigen from a cancer cell or tumor cell to induce a therapeutic or prophylactic anti-cancer effect in a vertebrate host, which involves the introduction of an antigenic composition containing the specified host into the specified host a cancer antigen or a tumor-associated antigen from a cancer cell or a tumor cell, and an effective adjuvant (immunostimulating) amount of a combination of (1) AOSR, or its derivative, or its analogue, and (2) a cytokine or lymphokine, or an agonist of said cytokine or lymphokine. 50. The method of increasing the ability of an antigenic composition containing a selected allergen to mitigate an allergic reaction in a vertebrate host, which involves the administration of an antigenic composition containing the specified allergen and an effective adjuvant (immunostimulating) amount of the combination (1) ACP to the specified host, or its derivative or analog and (2) a cytokine or lymphokine, or an agonist of said cytokine or lymphokine. " 51. An antigenic composition that contains a selected antigen from a molecule or a part thereof, representing a molecule C or a part thereof, which is produced by the host in an undesirable way, in an undesirable amount or location, so as to reduce such an undesirable effect by including an effective adjuvant ( immunostimulating) "» amount of the combination of (1) ASR, or its derivative, or analog and (2) cytokine or lymphokine, or agonist " of the indicated cytokine or lymphokine. 52. The antigenic composition according to claim 51, where the selected antigen is a polypeptide, peptide or fragment produced from an amyloid precursor protein or an antibody to it. - 53. Antigenic composition according to claim 52, where the selected antigen is AD peptide, which is an internal fragment with 39-43 amino acids of the amyloid precursor protein, or a fragment of AD peptide. eh! - ! 54. Antigenic composition according to claim 53, where the selected antigen is a peptide DD o oosh has the amino acid o o sequence: 42) lyar Alla bil be Mu Mi dyar Bek Siu tuk Ya3Zp chai Biz niv biv tsiv Peo chuai ve Zhe Aza biz Avr az b1u bach yap suv o s1iu liwa Hi 110 01Uu Ia Me Uai zu biu Uai chai ize dav yu (vBto hv zho: 6), 55. Antigenic composition according to claim 54, where AOR is 529. 60 56. A method of increasing the ability of an antigenic composition that contains a selected antigen from a molecule or part thereof, representing a molecule or part thereof that is produced by the host in an undesirable way, in an undesirable amount or location, to reduce such an undesirable effect by including an effective adjuvant ( immunostimulating) amount of a combination of (1) ACP, or its derivative, or analog and (2) a cytokine or lymphokine, or an agonist of said cytokine or lymphokine. 65 57. The method of increasing the ability of an antigenic composition to prevent or treat a disease characterized by the deposition of amyloid in a vertebrate host, which involves the introduction of a polypeptide, peptide or fragment produced from the precursor protein of amyloid or an antibody to it, and an adjuvant (immunostimulating) amount to the specified host a combination of (1) ACP, or its derivative, or analogue and (2) a cytokine or lymphokine, or an agonist of said cytokine or lymphokine. 58. The method according to claim 57, where the selected antigen is the Ag peptide, which is an internal fragment of 39-43 amino acids of the amyloid precursor protein, or a fragment of the AD peptide. 59. The method according to claim 58, where the selected antigen is an AD peptide having the amino acid sequence: yu yAvr ayiv bi: Ma ko Mia Avr Beh 41iu Tukh bis Chai Kiv niv yiv tsu pam Chai Tya ba viv bi Ar chai 91Uu Yayag dav uv F1Uu ia 16 116 y1Uu 120 mmEE Chai yiu 41u Uai chuai khiv az iz chBSHO TI shock b). 60. The method according to claim 59, where the AOR is 529. 61. An adjuvant composition containing a combination of (1) an aminoalkyl leucosamine phosphate compound (ACP), or its derivative or analog and (2) a cytokine or lymphokine, or an agonist of said cytokine or lymphokine. 62. The adjuvant composition according to claim 61, where ACP is used in the form of a stable oil-in-water emulsion. 63. The adjuvant composition according to claim 61, where the cytokine or lymphokine is selected from the group consisting of granulocyte-macrophage colony-stimulating factor and interleukin-12. 64. An adjuvant composition according to claim 61, which additionally contains a diluent or carrier. 65. Adjuvant composition according to claim 61, where the cytokine or lymphokine is a granulocyte-macrophage SC colony-stimulating factor. 66. Adjuvant composition according to claim 61, where the cytokine or lymphokine is interleukin-12. Gee) 67. Adjuvant composition according to claim 61, where AOR is 529. (Се) сч « (22) and - - с;» -I se) sh» d) 50 42) F) ime) 60 b5