EP1165142A2

EP1165142A2 - Lipid-based artificial particles inducing cell-mediated immunity

Info

Publication number: EP1165142A2
Application number: EP00912299A
Authority: EP
Inventors: Masanobu Sugimoto; Max Arella; Yasuhiro Furuichi
Original assignee: Sapientia Therapeutics Ltd; Agene Research Institute
Current assignee: Sapientia Therapeutics Ltd; Agene Research Institute
Priority date: 1999-03-25
Filing date: 2000-03-23
Publication date: 2002-01-02
Also published as: WO2000057919A3; AU3412500A; WO2000057919A2

Abstract

Artificial immunogenic particles (1), methods for producing the same and uses thereof are disclosed. The immunogenic particles (1) of the invention comprise a lipid-based matrix (3), glycolipids (5, 6) and peptide-lipid conjugates (7, 8) embedded into this matrix (3). These particles (1) are highly efficient in inducing cell-mediated immunity (CMI) when administered to a host. The immunogenic particles (1) of the invention can be used as immunostimulating agents (e.g. vaccines), and as immunotherapeutic agents for preventing and/or treating infectious diseases and cancers in which CMI responses play an important role.

Description

LIPID-BASED ARTIFICIAL PARTICLES INDUCING CELL-MEDIATED IMMUNITY

Background of the invention

1) Field of the invention

The present invention relates to immunogenic particles, methods for producing the same and uses thereof. The immunogenic particles of the invention comprise a lipid-based matrix, glycolipids and peptide-lipid conjugates embedded into this matrix. These particles are highly efficient in inducing cell-mediated immunity (CMI) when administered to a host. The immunogenic particles of the invention can be used as immunostimulating agents (e.g. vaccines) and as immunotherapeutic agents for preventing and/or treating infectious diseases and cancers in which CMI responses play an important role.

2) Description of the prior art

Immunity is roughly divided into two categories, humoral immunity and cell- mediated immunity (CMI). Humoral immunity renders its action through antibody molecules, which directly bind to target antigens of micro-organisms such as bacteria and viruses and inactivate these microbes. In humoral immunity, B-cells produce antibodies which bind to microbes and inactivate them. On the other hand, CMI, also called cellular immunity, is a specific immune response mediated by T-lymphocytes. More particularly, in CMI, cellular responses such as T- lymphocyte proliferation, activation of macrophages, induction of cytotoxic T-cells

(CTLs) and delayed-type hypersensitivity (DTH) T-cells, results in destruction or elimination of pathogens and modified-self somatic cells (like those infected with viruses) as well as malignant cells. DTH T-cells release various lymphokines including interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), which activate macrophages or directly suppress the proliferation of foreign microbes including viruses. Activated macrophages are known to effectively inactivate foreign intracellular micro-organisms like Mycobacterium. CTLs are also involved in the killing of modified-self somatic cells such as those infected with micro-organisms and viruses or the control and elimination of malignant cells.

A body of data now have accumulated which indicate that helper-T-cells determine whether humoral immunity or CMI is preferentially induced. In this respect, two kinds of helper-T-cells, Th1 and Th2 are known to play key roles. Th1 cells are now mainly defined by their production of cytokine IL-2, IFN-gamma and tumor necrosis factor (TNF). Th2 cells are mainly defined by their production of cytokines IL-4, IL-5, IL-6, IL-10 and IL-13. Th1 and Th2 cells selectively stimulate respectively humoral and CMI responses.

Humoral immunity and CMI play differential roles in protection. Neutralizing antibodies directly inactivate various viruses and bacteria. Generally, intracellular microbes such as Mycobacterium tuberculosis and retroviruses are extremely resistant to humoral antibodies. On the contrary, CMI responses are considered to play important roles in the protection against these intracellular microbes. As an example, the activation of macrophages by Th1 -cells is necessary to efficiently inactivate M. tuberculosis. Moreover, ample evidence exists showing that CMI responses play a major role in the protection against retroviruses such as human immunodeficiency virus (HIV) and bovine leukemia virus (BLV), and in the control of malignant cells.

Selective induction of either humoral immunity or CMI is now possible by employing different immunization protocols such as the route of immunization or adjuvant. Because antigens in vaccines and immunotherapeutic agents generally do not elicit sufficient immune responses by themselves for protection, adjuvants are used to augment immunogenicity of the antigen. Various kinds of adjuvants have been reported, but most of them have not been of practical use because of their significant side effects. The most common adjuvant used in human vaccines at present is alum adjuvant consisting of aluminum hydroxide. The alum adjuvant, however, is not able to induce a good CMI response although it can induce a fairly good humoral immune response. It is also noted that the simultaneous induction of humoral immunity with CMI sometimes abrogates the effect of CMI responses, and the induction of CMI responses alone is considered preferable for the vaccines and therapeutic agents for diseases in which CMI plays a major role.

Another important aspect of vaccines and therapeutic agents is that they should have minimal adverse side effects. The induction of humoral antibodies itself may be the cause of side effects such as allergy or the appearance of facilitating antibodies that could increase the seriousness of an infection. In order to avoid such side effects, it may be of important to induce CMI responses by vaccination without the appearance of a humoral response. Furthermore, vaccines and therapeutic agents which are preferably composed of natural material present in or tolerated by the body may avoid unexpected side effects.

In recent years, efforts have been devoted to the analysis of defined antigenic sites of many viruses, other infectious diseases and malignant cells. More specifically, for HIV-1 , the causative virus for acquired immunodeficiency syndrome (AIDS), many antigenic sites which elicit CMI (DTH, CTL and Th responses), have been identified and described in the literature. The identification of such sites is a prerequisite for the development of a potent CMI vaccine that may either bear or lack B-cell inducing epitopes. In immunology and vaccinal studies, it is common knowledge that CMI may be linked to short sequences of amino acids in a portion of a synthetic recombinant antigen or of a short synthetic peptide. This may induce a strong defense against infections. As an example, the T-cell antigenic sites from AIDS viral proteins are known (AIDS Th and CTL peptides, Los Alamos HIV Molecular Immunology Database, Los Alamos National Laboratory, Los Alamos, N.M., USA, 1996) and may constitute a basis for the selection of tailor-made vaccines from the analysis of AIDS virus carried by a particular individual and the selection of appropriate epitopes corresponding to such a virus. It is hypothesized that an ideal CMI antigen should facilitate each of the three main steps for raising a T-cell response, namely:

1) processing of the immunogenic particles by an antigen-presenting cell (APC) such as a macrophage or a dendritic cell; 2) presentation of the proper T-cell epitope as an exposed epitope to T-cells in conjunction with a class II Major Histocompatibility Complex (MHC) on the antigen presenting cells (APC) surface;

3) recognition of such a complex by a helper-T-cell receptor specifically identifying some combination of peptide and class II protein and initiating a T-cell response; and

4) similarly, recognition of such a complex by a CTL-T-cell receptor, specifically identifying some combination of peptide, and class I protein and initiating a T- cell response with the aid of helper T-cells.

It is also hypothesized that to be really effective, a CMI antigen should comprise carbohydrates. The induction of CMI is related to an evolutionary background in the immune system. It is conceivable that our body has refined several mechanisms to recognize the carbohydrate structure of various infectious agents and leading to the activation of immune system. The mannose receptors of macrophages may be one of such mechanism since they are well known to bind molecules containing non-reducing terminal carbohydrates with the following order of potency: D-mannose, L-fucose, D-GlcNAC (penta-N-acetylchitopentaose), D- glucose, D-galactose [Weigel, P. H., 1992 in «Glycoconjugates» (Allen, H.J. and Kisailus, E.C. Eds) Marcel Dekker, Inc. p 421-497]. It is also generally believed that these receptors are involved in inflammation, wound healing, antigen processing and other surveillance functions within the body. Finally, mannose residues are well reported stimulators of 11-12 release and activation of Th1 cells types.

U.S. patents Nos. 5,080,896; 5,552,141 ; 5,643,574; and 5,709,879 have attempted to provide compositions, immunogens and/or methods for immunizing a host. However, the idea behind these inventions is different from the present invention since these U.S. patents do not suggest the use of glycolipids, an essential element of the present invention for inducing a strong and effective CMI response.

The prior art has also attempted to provide CMI antigen by incorporating peptides (antigen) and glycolipids in a lipid-based matrix. For example, Noguchi et al. (J. Immunol. 146, 3599-3603, 1991) describe a liposome vaccine prepared by reconstituting proteins antigen into liposomes coated with mannan-cholesterol. Similarly, Sugimoto et al. (FEBS Letters, 363: 53-56, 1995) describe similar liposomes coated with oligomannose-dipalmitoyl phosphatidyl-ethanolamine.

These known systems have however the following defects:

1) The antigen is not conjugated with lipid, and the efficiency to reconstitute peptide antigens into liposome is highly variable depending on the peptide. For example, the reconstitution efficiency of free peptide is usually lower than that of peptide-lipid conjugate. It is especially difficult to efficiently incorporate hydrophilic peptides into lipid-based matrix as compared to hydrophobic peptides;

2) Proteins induce stronger humoral immune responses which could render side effects such as allergy or the appearance of facilitating antibodies;

3) Coating liposomes with glycolipids is a time-consuming and laborious process. Furthermore, non water-soluble glycolipids are not applicable to the method of coating liposomes.

Taking into consideration the background as mentioned above, there is a need for effective immunogenic particles devoid of the above mentioned defects. More particularly, there is a need for artificial immunogenic particles composed essentially of natural constituents and inducing a CMI response toward a particular peptide with a minimal or a total lack of humoral immunity. There is also a need for a simple method for preparing such artificial immunogenic particles and for vaccines and pharmaceutical compositions comprising such immunogenic particles.

SUMMARY OF THE INVENTION

It is a first object of the invention to provide artificial immunogenic particles comprising: a) a lipid-based matrix having an outer surface; b) glycolipids embedded into the matrix, the glycolipids having a lipidic portion and a saccharide portion; and c) peptide-lipid conjugates embedded into the matrix, the peptide-lipid conjugates having a lipidic portion and a peptide portion.

These immunogenic particles are able to induce a cell-mediated immune response against the peptide portion of the peptide-lipid conjugates when they are administered to a host in an immunogenic effective amount.

Preferably, the lipidic portion of at least some of the glycolipids is anchored into the matrix such that the saccharide portion of these anchored glycolipids is projecting at least partially from the outer surface of said matrix. Likewise, the lipidic portion of at least some of the peptide-lipid conjugates is anchored into the matrix such that the peptide portion of these anchored peptide-lipid conjugates is projecting at least partially from the outer surface of said matrix.

The particles of the present invention may be spherule-like particles, vesicles, liposomes or a mixture thereof, having a diameter varying from 10 nm to 50 μm, but more preferably from 0.1 μm to 10 μm.

These particles may be used directly as a vaccine or as an immunomodulator. It is also an object of the present invention to provide a pharmaceutical composition comprising such artificial immunogenic particles, and a pharmaceutically acceptable excipient. The pharmaceutical composition of the invention may further comprises anti-inflammatory agent(s) and/or compound(s) modulating immunity.

It is another object of the invention to provide a method for preparing lipid- based immunogenic artificial particles. This method comprises the steps of: a) preparing a mixture comprising: -an aqueous solution;

-lipids;

-glycolipids having a lipidic portion and a saccharide portion; and

-peptide-lipid conjugates having a peptide portion and a lipid portion; b) processing this mixture to yield a suspension containing particles comprising: i) a lipid-based matrix having an outer surface; ii) at least some of the glycolipids embedded into the matrix; and iii) at least some of the peptide-lipid conjugates embedded into the matrix.

The particles produced according to this method induce a cell-mediated immune response against the peptide portion when administered to a host in an immunogenic effective amount.

It is a further object of the invention to provide a method for inducing a cell- mediated immune response in a host, comprising administering to this host an immunogenic effective amount of artificial immunogenic particles. These particles comprise: a) a lipid-based matrix having an outer surface; b) glycolipids embedded into the matrix, the glycolipids having a lipidic portion and a saccharide portion; and c) peptide-lipid conjugates embedded into the matrix, the peptide-lipid conjugates having a lipidic portion and a peptide portion. Other objects and features of the invention will become apparent upon reading the following, non-restrictive description of several preferred embodiments thereof, made with reference to the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic cross-sectional view of the structure a single immunogenic particle according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to artificial immunogenic particles capable of preferentially inducing cell-mediated immunity (CMI), uses thereof and methods for preparing the same.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one ordinary skilled in the art to which this invention belongs.

As used herein, the terms "immunogen" (noun) and "immunogenic" (adjective) refer to any substance or molecule that generates an immune response when administered to a host.

Fig. 1 is a schematic view showing the structure of a single immunogenic particle according to the present invention. As stated herein before, each particle comprises: a) a lipid-based matrix 3 having an outer surface 4; b) glycolipids 5,6 embedded into the matrix 3; and c) peptide-lipid conjugates 7,8 also embedded into the matrix. The lipid-based matrix 3 is the major compound entering into the composition of the particles. The lipid matrix 3 constitute a carrier-like substance wherein immunogenic components (glycolipids 5,6 and peptide-lipid conjugates 7,8) are embedded as it will be explained in details herein after. The matrix 3 may be shaped such that the particles of the invention are spherule-like particles, vesicles and/or liposomes having a diameter varying from 1 nm to 50 μM, preferably from 0.1 μM to 10 μM.

As used herein, the term "lipid", in conjunction with the term "matrix", refers to any compounds, natural or artificial, which are soluble in organic solvents and are of a structural type which includes fatty acids and their esters, cholesterol, cholesteryl esters, and phospholipids. For example, natural lipids obtained from egg yolk soybean or from other vegetables or from animals may enter into the composition of the lipid matrix, including lipids which have been modified by increasing their degree of saturation by hydrogen addition. More specific examples include cholesterol, sterols such as gangliosides, phosphatidylethanolamines such as DPPE; phosphatidylcholines such as DPPC; phosphatidylserines such as

DPPS; phosphatidic acids such as DPPA; and other similar types of molecules bearing identical chemical properties. In fact, any natural lipid or other non-toxic chemical substance able to anchor immunogenic components such as glycolipids and peptide-lipid conjugates, is usable in the lipid matrix of the present invention.

As also shown in Figure 1 , the glycolipids 5,6 embedded into the matrix have a lipidic portion (5',6'; white) and a saccharide portion (5", dotted; 6", black), both portions being covalently conjugated one to the other. The glycolipids 5,6 are found throughout the lipid matrix 3. However, the glycolipids 5,6 are preferably embedded into the matrix 3 such that their lipidic portion 5',6' is anchored to the lipid matrix 3 and that their saccharide portion 5", 6" projects at least partially from the outer surface 4 of the lipidic matrix 3.

The fact that the saccharide portion projects from the outer surface of the matrix is important in order to be accessible to the immune system. Nevertheless this is not essential since it is believed that the saccharide portion of glycolipids buried into the matrix will become directly accessible upon processing of the particles by the immune system. It is also believed that the saccharide portion plays an important role as an adjuvant thereby increasing the induction of CMI responses towards the peptide portion of the peptide-lipid conjugates.

The saccharide portion of the glycolipids is preferably composed of oligosaccharides having 2 to 30 saccharide residues. Longer polysaccharides like mannan or others saccharides, polysacchrides and oligosacchrides containing mannose residues such as mannohexaose, mannopentaose and mannotriose are usable. Polysacchrides and oligosaccharides containing fructose residues or other sugar residues are also usable in the present invention.

For forming a complete glycolipid, these saccharides are conjugated with lipids (thereby forming the lipid portion) such as dipalmitoyl phosphatidyethanolamine (DPPE) and cholesterol or any other lipid as follows: natural lipids obtained from egg yolk soybean or from other vegetables or from animals may be used including those which have been modified by lowering the degree of unsaturation by hydrogen addition. Others examples include sterols such as gangliosides, cholesterol ; phosphatidylethanolamines such as DPPE; phosphatidylcholines such as DPPC; phosphatidyserines such as DPPS; phosphatidic acids such as DPPA; and other similar types of molecules bearing identical chemical properties.

Although the illustrated schematic example comprises two types of glycolipids, a person skilled in the art will easily understand that the type, size, and concentration of glycolipids found in the particles of the invention are selected to suit each particular purpose. Of course, a single type of glycolipids as well as many distinct types of glycolipids may be present in a single particle. Glycolipids are available commercially or can be prepared by a skilled in the art. For example, Mizuochi et al. (J. Biol. Chem. 1989:264; 13834-13839) describe a method for preparing M5-DPPE. As also shown in Figure 1 , the peptide-lipid conjugates 7,8 embedded into the matrix 3 have a lipidic portion (7', 8'; white) and a peptide portion (7", dotted; 8", black), both portions being covalently conjugated one to the other. The peptide- lipid conjugates 7,8 are found throughout the lipid matrix 3. However, the peptide- lipid conjugates 7,8 are preferably embedded into the matrix 3 such that their lipidic portion 7',8' is anchored into the lipid matrix 3 and that their peptide portion 7", 8" projects at least partially from the outer surface 4 of the lipidic matrix 3.

The fact that the peptide portion projects from the outer surface of the matrix is important in order to be accessible to the immune system. Nevertheless this is not essential since it is believed that the peptide portion of peptide-lipid conjugates buried into the matrix will become accessible upon processing of the particles by the immune system. The peptide portion of the peptide-lipid conjugates act as immunogen of helper T-cells and cytotoxic T-cells to which a CMI response is elicited.

The term "peptide" herein includes any natural or synthetic compounds containing two or more amino acids. Therefore, it comprises proteins, glycoproteins, and proteins fragments derived from pathogenic organisms such as viruses, bacteria, parasites and the like, or proteins isolated from normal or pathogenic tissues, such as cancerous cells. It also includes proteins and fragments thereof produced through recombinant means. Preferably, peptides having 8 to 25 amino acids are used. However, longer peptides and proteins with more than 25 amino acid residues are also usable. Peptides are chosen accordingly to the desired use. In the case wherein the peptide comprises sugar moieties, the CMI response could be also directed against some of said sugars.

For example, HIV-related peptide(s), and fragments thereof, may be used for eliciting a immune response and/or immunity towards the HIV virus. Examples of known* HIV-related peptides includes:

- DRWEKIRLRPGGNKKYK; -HIVWASRELERFAVN; - PIVQNIQGQMVHQAISPRTL; - GPKEPFRDYVDRFYKTLRAE;

- QLLFIHFRIGCRHSR; - IGQHRTKIEELRQHL;

- NFKRKGGIGGYSAGE; - NVWATHACVPTDPNPQEWLE;

- VEQMHEDIISLWDQSLKPC; - HEDIISLWDQSLKPC; - EWIRSANFTDNAKT; - RIHIGPGRAFYTTKN; and

- RIQRGPGRAFVTIGK.

*(Los Alamos HIV Molecular Immunology Database, Los Alamos National Laboratory, Los Alamos, N.M., USA, 1996)

Hepatitis B and C, HPV, HSV, EBV and influenza are other viral infections wherein the particles of the invention could be useful.

Similarly, malignant cell-related peptide may be used for inducing a therapeutic immune response towards cancerous cells. In fact, any peptide or polypeptide, natural or synthetic, corresponding to an antigenic site of a pathogenic infectious agent and or pathogenic cell can be usable as an immunogen.

For example, synthetic melanoma antigens such as Mage-2, Mage-3, Mart, gp 100, and tyrosinase could be candidate peptides for therapeutic cancer vaccines. Other tumor rejection antigenic peptides to be considered include HER-

2-NEU (ovarian and breast cancer), PSA (prostate), MUC-1 (breast, colo-rectal, lung), HPV-16 and HPV-18 (cervical), p53, CEA, etc.

Any natural lipid or other non-toxic chemical substance that may be conjugate to a peptide may be usable in the present invention. In preferred embodiments, cholesterol and myristic acid is used. Others examples includes: natural lipids obtained from egg yolk soybean or from other vegetables or from animals may be used including those which have been modified by lowering the degree of unsaturation by hydrogen addition. Further examples include sterols such as gangliosides; phosphatidylethanolamines such as DPPE; phosphatidylcholines such as DPPC; phosphatidylserines such as DPPS; phosphatidic acids such as DPPA; and other similar types of molecules bearing identical chemical properties. Phosphorylcholine and dodecylamines as having appeared in the literature are also usable (Jang, Y.S., Lim, K.H. and Kim, B.S.; Eur. J. Immunol. 1991 , 21 : 1303-10; Singh, S.B. and Leskowitz, S.; J. Immunol. 1978, 120:734-8). Peptide-lipid conjugaison is performed according to techniques known in the art. A specific example of conjugation is provided hereinafter (see Example 1).

Although the illustrated schematic example comprises two types of peptide- lipid conjugates, a person skilled in the art will easily understand that the type, size, and concentration of peptide-lipid conjugates found in the particles of the invention are selected to suit each particular purpose. Of course, a single type of peptide-lipid as well as many distinct types peptide-lipid conjugates may be present on a single particle. A particle having many distinct types peptide-lipid conjugates will thereby function as multivalent immunogen.

The main advantages of using peptide-lipid conjugates is that they are incorporated more efficiently in the lipidic matrix than free peptides, and that the antigenicity of peptide-lipid conjugates is comparable to or sometimes even higher than free peptides as it will be shown in the following examples. Using glycolipids is also a major advantage since it is assumed that the glycolipids enhances CMI responses directed to the peptide since the surface of various infectious agents such as viruses, bacteria, yeast and protozoa is covered with carbohydrate and/or lipid moieties including those containing an high level of mannose and it is well known that the immune system employs these chemical structures for its efficient activation of a cell-mediated response.

The immunogenic particles of the invention are thus suitable as vaccines, immunomodulator, and/or as a therapeutic agent for infectious diseases and malignant tumors in humans and animals since CMI responses play an important role in protection and therapy of these diseases. Furthermore, since the immunogenic particles of the invention are constituted essentially of materials present in human and animal cells, it is expected that negative side effects are minimal once administered into a host.

The particles may also comprise therapeutically active agents such as anti- inflammatory agents, compounds modulating immunity, growth factors, nucleic acids (DNA, anti-sense, RNA) and/or antibodies for specifically addressing the delivery of the particles and/or to avoid toxicity.

Examples of anti-inflammatory agents comprise anti-cyclooxygenase (Cox- 2) inhibitors, steroids as well as DMARD agents (disease modifying anti-rheumatic drug). Examples of immunomodulating agents include interferons, interleukins, chemokines, etc. More specific examples comprise macrophage inflammatory proteins (MIP-1α, MIP-1 β, RANTES), Exodus-2 and 3, MCP-1 , MDC, activating cytokines (TNF-α, INF-γ, IL-2, IL-12, IL-15, IL-18) and inhibitory cytokines (IL-10, IL-4, IL-1).

A first non-restrictive specific example of a therapeutic use is the treatment of cancer. Obviously, in such case, the peptides embedded in the lipid matrix would be selected amongst the tumor specific antigens. Furthermore, the particles of the invention would further preferably comprise: 1) tumor specific antibodies (e.g. anti-HER2-NEU, anti-CD20, anti-MUC, etc.) and potent cytotoxic drug or toxin that will be delivered in/and around tumor cells; 2) growth factors or other cytokines that will up modulate class II HLA and/or tumor antigen expression. This in turn will allow a better efficacy of both HLA and cancer antigen recognition (immunization step) as well as tumor cell killing (cytotoxic efferent step); 3) activators of dendritic cells (e.g. IL-13) that will trigger anti-tumor specific cytotoxic T-cell response.

A second non-restrictive specific example of a therapeutic use is the treatment of rheumatoid arthritis (RA). In such case the particles would be covered with specific monoclonal antibodies (MAb) and would also comprise anti- inflammatory agents. The Mab would be specific for periarticular and/or synovial membrane antigens and/or chondrocytes. This strategy would allow delivery to the disease site of a high concentration of drug while avoiding systemic deleterious side effects. A similar strategy could be used in transplantation of cells, tissue, and organs allowing delivery to the transplant of high concentrations of steroids, inhibitory cytokines and/or inhibitory growth factors (e.g. TGF-β).

It is also believed that the particles of the invention could be used as a down regulator (immunomodulator) of inflammatory response. To do so, the particles would comprise peptides known to normally stimulate Th1 cells, in order to reduce a CMI Th2 response towards these "inflammatory peptides". This "switch" would be particularly interesting in the treatment of arterial sclerosis (see Bachmaier et al., Science, 283: 1335-9) and in the prophylaxis of graft rejection.

Obviously, the immunogenic particles of the invention may be administered alone or as part of a pharmaceutical composition. The immunogenic particles and/or pharmaceutical compositions of this invention, or those which are manufactured in accordance with this invention, may be administered by any suitable route.

For example, mucosal surfaces are suitable for the administration of the particles of the invention. Ocular (conjunctival, corneal), oral (buccal, sublingual, perlingual, ingestion), vaginal, nasal, pulmonary, gastrointestinal and rectal are other examples of suitable routes of administration.

More particularly, the particles of the invention and/or pharmaceutical compositions comprising the same may be given orally in the form of tablets, capsule, powders, syrups, etc., or nasally by means of a spray. They may also be formulated as creams or ointments. They may be formulated as drops, or the like, for administration to the eye. They may also be given parenterally, for example intravenously, intramuscularly or sub-cutaneously by injection or by infusion. For preparing such compositions methods well known in the art may be used. For example, oral administration may necessitate the use of a gelatin and/or enterocoated capsule to make sure that the composition is not digested in the stomach. Any pharmaceutically acceptable carriers, diluents, excipients, or other additive usually used in the art, are suitable depending upon the desired method of administering it to a patient. For injectable solutions, excipients which may be used include, for example, water, isotonic saline solution, alcohols, polyols, glycerine, and vegetable oils.

The pharmaceutical compositions of the invention may also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts, buffers, coating agents or antioxidants.

The pharmaceutical compositions of the invention may also contain other therapeutically active agents such as anti-inflammatory agents, compounds modulating immunity, growth factors and nucleic acids as described herein before.

The amount of particles to be administered is an immunogenic and/or therapeutically effective amount. An immunogenic and/or therapeutically effective amount of particles is that amount necessary for inducing an effective immune response towards the peptide(s) and/or the sugar moieties of the particles of the invention once these particles have been administered to a host.

Suitable dosages will vary, depending upon factors such as the amount of peptide-lipid conjugates and glycolipids embodied in the particles, the disease or disorder to be treated, the route of administration and the age and weight of the individual to be treated. Without being bound by any particular dosages, it is believed that for instance for parenteral administration, a dosage of from about 0.01 to about 0.1 mg/kg of particles comprising about 0.5% to 5% of glycolipid and about 0.05% to 0.5% of peptide-lipid conjugates, for a first injection, and a dosage of from about 0.01 to about 0.05 mg/kg for a second injection may be suitable for inducing a strong and effective CMI response in that individual. This dosage may be repeated as often as appropriate. Typically administration may be 2 times at 10-14 days interval. If side effects develop, the amount and/or frequency of the dosage can be reduced. A typical unit dose for incorporation into a pharmaceutical composition would thus be preferably about 5 mg of particles, suitably between 1 and 20 mg.

The lipid-based immunogenic particles of the invention may be easily prepared by a simple method which comprises the steps of: a) preparing a mixture comprising: an aqueous solution, lipids, glycolipids and peptide-lipid conjugates; b) processing said mixture to yield a suspension wherein at least some of the glycolipids and peptide-lipid conjugates are embedded into the lipid-based particles as explained herein before.

More particularly, at first, the three components entering into the immunogenic particles of the inventions, viz. the binding lipids (as the future lipid matrix), the peptide-lipids and the glycolipids are well mixed together by a mortar, a mixer or a vortex before being further mixed into an aqueous solution such as phosphate buffered saline (PBS). Obviously, the three components may also be mixed directly into the aqueous solution. Freeze-thaw cycles (two or more) are also effective to mix well the components.

The mixing temperature is preferably varying between 40°C and 60°C, and more preferably it is about 50°C. The mixing temperature is selected in order to be above the gel-liquid crystal transition temperature of the lipids of the mixture entering into the matrix.

Preferably, the mixture is then extruded through the filter (membrane) of an extruder leading to an emulsion comprising micro-sized particles. In a preferred embodiment, the mixture is extruded using The Extruder™ (Lipex Biomembranes

Inc., Vancouver, Canada). Others devices and methods may be suitable to prepare the immunogenic particles of the invention provided that the resulting emulsion comprise micro-sized particles. Such suitable devices includes fast- vortex and ultrasound.

For the extrusion step, the pore size of the filter is selected according to the procedure to be used for the administration of the final product. In the preferred embodiment a filter having pores of 0.6 μm to 1.0 μm is used, leading to particles having a diameter varying from 0.6 μm to 1.0 μm which are suitable for parenteral administration.

A person skilled in the art will understand that other therapeutically active agents such as anti-inflammatory agents, compounds modulating immunity, growth factors and nucleic acids may also be added to the mixture during the preparation of the particles. In other situations and as explained herein before, like for the development of an oral vaccine, it might be preferable to incorporate the final suspension of immunogenic particles in gelatin capsules or other types of known enterocoatings to prevent their exposure to gastric fluids.

No microscopic studies have been done to verify the exact structure of the particles prepared according to the above described method. However, a person skilled in the art will understand that various types of particles may be suitable, as well as various methods for producing the same. For example, various types of lipid particles embodying peptide-lipid conjugates and glycolipids such as spherule-like particles, vesicles and liposomes may be easily be prepared by various methods known to those skilled in the art. Thus, such spherules, vesicles, liposomes and others similar types of lipid-based particles and methods for producing the same are all considered to be within the scope of the present invention if they comprise peptide-lipid conjugates and glycolipids embedded in a lipid-based matrix.

As it will now be demonstrated by way of examples hereinafter, the particles of the present invention possesses an activity to induce a strong CMI response against the peptide contained therein when they are administered in an immunogenic effective amount. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

EXAMPLES

Experiments were performed to show that it is possible to induce CMI based on selected epitopes (peptides) embedded into the particles of the invention. As an example and a proof-of-concept, HIV-1 epitopes were selected to show the immunogenicity of the preparations in a mouse model.

Preliminary studies were first performed in order to select a model antigen. The immunogenicity of 12 HIV peptides was compared in Balb/c mice. It was found that a peptide with the amino acid sequence QLLFIHFRIGCRHSR, named herein Sap9, had the strongest immunogenicity to induce both a DTH response and a lymphocyte proliferation response (results not shown). Therefore, the Sap9 peptide was chosen as a model HIV peptide and used as a model antigen in the subsequent experiments (see Examples 1 and 2). Sap7, a peptide, having the sequence TRKSIRIKRGPGRAF, was selected as a negative control since it showed virtually no immunogenicity both in the footpad DTH response and in the lymphocyte proliferation response. It is also noted that footpad DTH responses and lymphocyte proliferation responses paralleled each other, implying that these two markers are usable as an interchangeable indicator of CMI. Using an ELISA immunoassay failed to show a humoral response to the individual peptides.

Example 1 : Preparation of immunogenic particles containing Sapθ and mannan-cholesterol and their immunogenicity in Balb/c mice. 1) Preparation of the immunogenic particles of the invention

PGL1 : PGL1 is a preparation of particles having only glycolipids embodied into a lipid matrix. PGL1 was prepared and tested as a negative control vaccine. Two hundreds mg of lipids (comprising one part of cholesterol and two parts of DPPC in molar), and 50 μg mannan-cholesterol (Dojindo Laboratories, Kumamoto, Japan) were vortexed in 1 ml phosphate buffered saline (PBS) (pH 7.2). The mixture was incubated at 50°C for 10 min then submitted to an extrusion at 50°C by using The Extruder™ (Lipex Biomembranes Inc., Vancouver, Canada). A polycarbonate filter (Nucleopore™, Costar, Cambridge, MA), having 1 μm diameter's pores, was used during the extrusion procedure which was repeated five times.

PGL2: PGL2 is a preparation of particles having only peptide-lipid conjugates embodied into a lipid matrix. PGL2 was prepared as for PGL1 except that 5 mg of Sap9/Myr, (the Sap9 peptide conjugated with myristic acid) was used to prepare the mixture instead of mannan-cholesterol (PGL1).

Conjugation of Sap9 with myristic acid was performed during the synthesis of the Sap9 peptide accordingly to standard procedures (Sheppard et al. J. Amer. Chem. Soc, (1975) 97:22, p 6554; Sheppard et al. J. Chem. Soc Chem. Comm., (1979), p 423; Sheppard et al. J. Chem. Soc, (1981), p 529-537). Briefly, before cleaving the synthesized peptide out of the synthesizer's column, an extra step was performed wherein a myristic acid solution (2 ml of 100 mM) was added manually during the amino acid addition step. Then, the myristilated peptide was allowed to cleave and purified as normal.

PGL3: PGL3 is a preparation of whole artificial immunogenic particles according to the invention. PGL3 particles had both glycolipids and peptide-lipid conjugates embodied into a lipid matrix. PGL3 was prepared as for PGL1 and

PGL2 except that both 50 μg mannan-cholesterol and 5 mg Sap9/myr were used to prepare the mixture. Once prepared, the amounts of cholesterol and carbohydrate found in the particles of the various PGLs preparations were respectively determined using the assay kit of Cholesterol Cll-Test™ (Wako Purechem. Inc., Ltd.) and the anthrone- sulfate method (D.L.Morris, Science 107, 254, 1948). The amount of peptide in the particles was determined by a standard protein assay kit (Bio-Rad) after dissolution of the particles in 2% sodium dodecyl sulfate solution by heating at 98 °C for 30 min.

2) Assessment of a DTH footpad response in Balb/c mice.

7-week old female inbred Balb/c mice were obtained from Charles River Japan Inc. (Yokohama). They were divided into three groups; group 1 (6 mice) received PGL1 inoculation, group 2 (5 mice) received PGL2 inoculation and group 3 (7 mice) received PGL3 inoculation. Mice were inoculated subcutaneously on day 0, with 0.5 μg/50 μl of PGL1 , PGL2 or PGL3 at two positions of the lumber region, and on day 7 the same mice were inoculated with the same PGL preparation in the nuchal region. Seven days after the second inoculation, a DTH response was assessed by footpad swelling response as follows: mice were challenged subcutaneously in the right hind footpad with 20 μg Sap9/myr incorporated into 25 μg alum adjuvant in 25 μl PBS, and in the left footpad with the same solution without Sap9/myr as a control. The footpad swelling response was assayed 24 hours after the challenge. The specific footpad swelling responses were expressed as the difference between the thickness of right and left footpad. Footpad thickness was measured by a Peacock™ dial thickness gauge (Osaki MGF Inc., Ltd., Tokyo).

3) Results

Table I shows the recovery of each component in the various preparation of particles. The recovery of total lipids was between 50% and 60% and that of mannan-cholesterol was 78-98%. On the other hand, the recovery of Sap9/myr was low in this experiment (2-7%). However, the recovery of Sap9/myr was much higher when mannan-cholesterol was also added to the mixture during the preparation of the particles: PGL2 (without mannan-cholesterol) = 2%; PGL3 (with mannan-cholesterol) = 7%.

Table II shows DTH responses induced by PGL1 (group 1), PGL2 (group 2) and PGL3 (group 3). Only the mice inoculated with PGL3 containing both Sap9/myr and mannan-cholesterol induced a strong DTH response. It is noted that a relatively small amount of mannan-cholesterol (0.32 μg/mg total lipids) and peptide-lipid conjugates (2.88 μg/mg total lipids) was enough to render an adjuvant effect (see Table I).

Example 2: Preparation of particles containing Sap9 and M5-DPPE and their immunogenicity in Balb/c mice

1) Preparation and the immunogenic particles of the invention and recovery of each component

Three others preparation of particles, PGL4, PGL5, PGL6, respectively similar to PGL1 , PGL2 and PGL3 were made in order to compare the adjuvant effect of a second type of glycolipid, viz. mannopentaose-DPPE (M5-DPPE), and also compare the immunogenic efficiency of peptide-lipid conjugates vs simple non lipid-conjugated peptides.

The procedure for the preparation of PGL4, PGL5 and PGL6 was essentially the same with that for PGL1 , 2 and 3 except for the constituents of the various mixtures. PGL4 contained the matrix alone, PGL5 contained Sap9 with M5-DPPE, and PGL6 contained Sap9/myr with M5-DPPE. M5-DPPE was purchased from Dojindo Laboratories (Kumamoto, Japan). 2) Recovery of each component

Table III shows the recovery of each of the components in the preparations of PGL4, PGL5 and PGL6. The recovery of both M5-DPPE and Sap-9 peptide was higher in the PGL6 particles using its derivative conjugated with myristic acid (Sap9/myr) than in PGL5 using the non-conjugated Sap9 peptide. This suggest that simultaneous presence of both peptide-lipid and glycolipid enhances the incorporation of both components into the lipid-base particles.

3) Immunization protocol

Balb/c mice (five to seven mice for each group) were immunized subcutaneously 2 times at 14-day intervals with 0.2 ml of PGL4, PGL5 or PGL6 on days designated as day 0 and day +14. The mice were bled from the retro-orbital sinus at day -1 (pre-bleed) and on the day of termination (day +28) for a minimum volume of about 6 ml/Kg. As a positive control, 10 μg of the recombinant protein HIV gp160 (Bartels Inc., Issaquah WA 98027), was injected intraperitoneally to four Balb/c mice with Freund's Complete Adjuvant on day designated as day 0, followed by a second 10 μg dose with Freund's Incomplete Adjuvant on day +14. The mice were bled at weekly intervals after day +14 until termination of the experiment and the ELISA titer determined. The mice of the experimental and control groups were assayed for serum antibody and lymphocyte proliferation response. The results of which are reported hereinafter. The details of the schedule of immunization are given in Table V.

4) Assay of serum antibody immunity

The antibody responses to the single peptide (Sap9) and a control peptide

(Sap7) were assayed by enzyme linked immunoassay (ELISA). Each assay included a positive control serum (the anti gp160 antiserum obtained by the injection of a recombinant polypeptide of the whole protein) and negative control sera to ensure specificity of the assay for anti-peptide responses. During the ELISA assay for antibodies to the peptides, the sera were initially screened at ten fold dilutions (10x) starting from a dilution of 1/10 and subsequently at twofold dilutions starting at a dilution of 1/50. The upper 95% confidence limit of control data was used as the basis for identifying significantly different observations (mean + 1.96 standard deviates).

To ensure consistency of the assay results, all serum samples were frozen at - 20°C as acquired and assayed in a single group against the specific peptides. Assays were done in triplicate and repeated as required to verify the accuracy of the results. The ELISA assays were performed as indicated in Table V.

5) Assay of cellular immunity (CMI)

The mice were killed by exsanguination under isothane anesthesia and lymph nodes were obtained for assessment of CMI by measuring peptide induced T-cell proliferation responses in vitro. After removal of the lymph nodes, tissue biopsies of all injection sites and granulomas were submitted for microbiological assessment. The assay of all 30 mice was performed over 3 days in which 2 from each of the four immunized groups plus 2 non-immunized control mice were assayed on each day. Each assay of cell mediated immunity was performed as scheduled on Table V.

6) Results of the evaluation of vaccination, ELISA assay and lymphocyte proliferation response:

a) ELISA assay of the vaccinated mice

The results showed that none of the mice immunized with the various PGL preparations showed significant levels of antibodies (data not shown) while a small group of mice immunized by the recombinant gp160 antigen produced specific antibody titers against the same polypeptide and did react weakly with the Sap-9 peptide. This suggests that the PGL preparations with peptides failed to induce an antibody response.

b) Cell mediated immunity of the vaccinated mice

Table IV shows the results of mice immunized with PGL4, PGL5 or PGL 6 preparations of particles. Both the mice immunized with PGL5 and PGL6 induced moderate lymphocyte proliferation responses specific to Sap9. Both complete preparations of particles (PGL5 and PGL6) comprising glycolipids and peptide- lipids induced statistically significant responses. PGL6 preparation comprising the peptide-lipids further conjugated with myristic acid, induced the strongest responses. Therefore, the particles of the invention comprising both glycolipids and peptide-lipids conjugates are considered to be the most capable of inducing biologically meaningful CMI responses.

Although not shown, none of the serum samples of immunized mice showed a detectable antibody response against Sap9 or gp160 used as a positive control suggesting that the PGL complex is a poor or non-inducer of a humoral immune response.

Example 3: Assay of toxicity of the lipid-based artificial particles

A) Methodology:

An evaluation of the possible toxicity of the glycolipids complexes (GL) was undertaken in a mice model under two routes of administration. These experiments were conducted in order to assess the possible toxicity of the carrier molecules (PGL1) of the lipid-based artificial particles of the invention. Only the PGL1 complex was tested since it is generally accepted that the peptide-epitopes used as a specific vaccine are generally not linked to any toxic effects. 1) Injection of lipids

Two glycolipids complexes (GL) preparations respectively containing either 10 mg/ml and 40 mg/ml of PGL1 particles prepared as described previously and a control solution without lipid were used for the analysis of their toxicity in Balb/C female mice. Each preparation was injected either intravenously or subcutaneously into 6 mice per group for a total of 6 groups or 36 mice in all (Table VI). The mice were injected 2 times at 14 days intervals with the GL preparations containing either saline or PGL1 on days 0 and 14. Before injection, specimens were also tested for standard microbial sterility.

2) Assay ofHematology and Blood Biochemistry

Blood specimens were obtained from the mice for hematology assessment consisting of combined blood cell counts (CBC), differential counts (DIF), hemoglobin in g/L blood, hematocrit in IJL, mean corpuscular volume in fL (MCV), mean corpuscular hemoglobin per erythrocyte in pg (MCH) and mean corpuscular hemoglobin content in g/L (MCHC). Three mice for each group were bled from the saphenous vein under isothane anaesthesia at day 10 for a volume of about 6 ml/Kg. Half of the mice were exsanguinated on days of termination at day 21 and the other half at day 35. Blood samples were submitted for biochemical analysis for each group. The profile of assays were selected to examine liver and renal function in the mice. A detailed description of the various assays performed for each experimental group is presented in Table VII.

3) Assay of Tissues for Histopathology At the time of termination days 21 and 35, tissues specimens were obtained from spleen, liver, lung, and mesenteric lymph nodes. For the subcutaneously injected mice, the local lymph nodes were also examined after being processed for histology and sectioning and stained with hematoxylin and eosin.

4) Statistical analysis

The statistical analysis was performed in two stages. First, the subcutaneous (3 mice) and intravenous group (3 mice) were compared for significant differences. Where the groups were not statistically different, they were pooled for subsequent analysis comparing the effects of injection of 1 mg of lipid (6 mice) or 4 mg of lipid (6 mice) to the control group for the data obtained at days 10, 21 and 35. The student T test was used for these paired comparisons and a one tail 5% level of significance was the minimum criterion to disprove the null hypothesis.

B) Results

a) Toxicitity

The standard GL preparations injected sub-cutaneously into groups of mice using the same protocol as for previous experimental assessments of their use as a vaccine vehicle showed no signs of toxicity. Increasing the dose to 4 times the normal subcutaneous injection had no adverse effect on the mice, either immediately or delayed. There was no immediate or delayed effect observed over a period of 35 days for either 1 or 4 mg of PGL1 administered intravenously. Also, none of the mice used in the experimental groups showed any significant tissue changes or signs of toxicity measured by hematology and blood biochemistry evaluation. This trial showed that the PGL1 has no inherent subcutaneous or intravenous tissue toxicity for mice. More detailed analysis of the results is given in detail in the following paragraphs.

b) Microbiology sterility

No significant contamination was found associated with the GL preparations. None of the mice died as a results of the injections and none developed any significant granulomatous or abscesses.

c) Hematology results

Terminal bleeding of mice on days 21 and 35 was performed for evaluation of hematology and biochemistry assays. Although some minor variations were observed, none of the groups consistently showed any significant deviation from the hematological values obtained from the control group of mice which also generally agreed with published values.

d) Biochemistry results

Terminal bleeding of mice via the jugular vein on days 21 and 35 was used for all the biochemistry assays. Although some minors variations were observed, none of the groups consistently showed any significant deviation from the biochemical values obtained from the control group of mice which also generally agreed with published values.

e) Histopathology results

Virtually all of the tissues specimens from the s/c or i/v injected mice were evaluated as normal. Only the mice injected at 4 mg of the PGL1 preparation showed mild white pulp hyperplasia in one of the mice and mild follicular hyperplasia in the mesenteric lymph nodes in 4 of the 6 mice sacrificed at days 21 and 35. These changes were assessed as not significant by a pathologist.

Of course, numerous modifications and improvements could be made to the embodiments that have been disclosed herein above. These modifications and improvements should, therefore, be considered a part of the invention, the scope of which is to be determined by the following claims.

Table I: Recovery of each of the components in the PGL preparations of Example 1

The amount of total lipids was estimated by determining the amount of cholesterol. The amounts of mannan-cholesterol and of Sap9/myr were estimated by determining the amounts of sugar and peptide as described in the text.

The percentage recovery is calculated as the amount of component recovered per initial amount of each material.

Table II: Immunogenicity of the PGL preparations of Example 1 in Balb/c mice (DTH footpad swelling responses)

Group 2 or group 3 was compared with group 1.

Table III: Recovery of each of the components in the PGL preparations of Example 2

The amount of total lipids was estimated by determining the amount of cholesterol. The amounts of M5- DPPE and of Sap9 and Sap9/myr were estimated by determining the amounts of sugar and peptide as described in the text.

The percentage recovery is calculated as the amount of component recovered per initial amount of each material. 30 mg M5-DPPE and/or 5 mg of Sap9 or Sap9/myr were added to 100 mg lipid. Table IV: Immunogenicity of the PGL preparations of Example 2 in Balb/c mice (Lymphocyte proliferation responses)

Statistics were analysed between the lymphocyte proliferation responses with a stimulating (Sap 9) and those with a control antigen (Sap 7).

The value 13.15 was eliminated by Chauvenet criterion.

Table V: Schedule of immunization of the various preparations used

CMI assay is performed by a lymphocyte proliferation assay and a cellularity analysis of the lymph nodes of the vaccinated animals

Table VII: Groups of mice and protocol for toxicity testing of the PGL1 preparation

* Description of tests and abbreviations used in Table VII:

Total tests

L= Liver profile 54

H= Hematology (CBC/Differential) 54

T= Tissue histology (Injection site, Spleen, Liver, Lung,

Mesenteric lymph nodes). Four blocks/mouse @ 3 tissues/block 72 M= Microbiology (Aero/Anaero). Injected material (GL) and tissues 6 P= Pathology evaluation of slides 72

Claims

CLAIMS:

1. Artificial immunogenic particles (1) characterized in that they comprise: a) a lipid-based matrix (3) having an outer surface (4); b) glycolipids (5,6) embedded into said matrix, said glycolipids (5,6) having a lipidic portion (5',6') and a saccharide portion (5", 6"); and c) peptide-lipid conjugates (7,8) embedded into said matrix, said peptide-lipid conjugates having a lipidic portion (7', 8') and a peptide portion (7", 8"); said particles (1) inducing a cell-mediated immune response against said peptide portion (7", 8") when administered to a host in an immunogenic effective amount.

2. The particles of claim 1 , characterized in that the lipidic portion (5', 6') of at least some of said glycolipids (5,6) is anchored into said matrix (3) such that the saccharide portion (5", 6") of said anchored glycolipids (5,6) is projecting at least partially from the outer surface (4) of said matrix (3) ; and in that the lipidic portion (7', 8') of at least some of said peptide-lipid conjugates (7,8) is anchored into said matrix (3) such that the peptide portion (7", 8") of said anchored peptide-lipid conjugates (7,8) is projecting at least partially from the outer surface (4) of said matrix (3).

3. The particles according to claim 1 or 2, characterized in that said particles (1) are spherule-like particles, vesicles, liposomes or a mixture thereof.

4. The particles according to any one of claims 1 to 3, characterized in that said lipid-based matrix (3) comprises lipids selected from the group consisting of gangliosides, cholesterol, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, phosphatidic acid, and mixture thereof.

5. The particles according to any one of claims 1 to 4, characterized in that said lipid portion (5', 6') of said glycolipids (5,6) is selected from the group consisting of cholesterol, gangliosides, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidic acids.

6. The particles according to any one of claims 1 to 5, characterized in that said saccharide portion (5", 6") is selected from the group consisting of polysacchrides or oligosacchrides comprising mannan, mannohexaose, mannopentaose, mannotriose and or fructose residues.

7. The particles according to any one of claims 1 to 6, characterized in that said lipid portion (7', 8') of said peptide-lipid conjugates (7,8) is selected from the group consisting of cholesterol, myristic acid, gangliosides, phosphatidylethanolamines, phosphatidylcholines, phosphatidyl-serines, and phosphatidic acids.

8. The particles according to any one of claims 1 to 7, characterized in that said peptide (7",8") comprises sugar moieties, and in that said cell-mediated response is directed against at least some of said sugars.

9. The particles according to any one of claims 1 to 8, characterized in that said peptide (7", 8") is selected from the group consisting of viral-related peptides, malignant cell-related peptides or inflammatory related peptides.

10. The particles according to claim 9, characterized in that said viral-related peptide is a HIV-related peptide selected from the group consisting of :

- DRWEKIRLRPGGNKKYK;

- HIVWASRELERFAVN; - PIVQNIQGQMVHQAISPRTL;

- GPKEPFRDYVDRFYKTLRAE; - QLLFIHFRIGCRHSR;

- IGQHRTKIEELRQHL;

- NFKRKGGIGGYSAGE; - NVWATHACVPTDPNPQEWLE;

- VEQMHEDIISLWDQSLKPC; - HEDIISLWDQSLKPC;

- EWIRSANFTDNAKT;

- RIHIGPGRAFYTTKN;

- RIQRGPGRAFVTIGK; and fragments thereof.

11. The particles according to claim 9, characterized in that said malignant cell- related peptide is selected from the group consisting of Mage-2, Mage-3, Mart, gp 100, tyrosinase, HER2-NEU, PSA, MUC-1 , HPV-16, HPV-18, p53 and CEA.

12. The particles (1) according to claim 3, characterized in that they have a diameter varying from 10 nm to 50 μm.

13. The particles (1) according to claim 12, characterized in that they have a diameter varying from 0.1 μm to 10 μm

14. The particles according to any one of claims 1 to 13, characterized in that they further comprise at least one compound selected from the group consisting of anti-inflammatory agents, immunomodulating agents, growth factors, nucleic acids and antibodies.

15. A vaccine or an immunomodulator comprising immunogenic particles according to any one of claims 1 to 14.

16. A pharmaceutical composition, characterized in that it comprises artificial immunogenic particles according to any one of claims 1 to 14, and a pharmaceutically acceptable excipient.

17. The pharmaceutical composition according to claim 16, characterized in that it comprises at least one compound selected from the group consisting of anti- inflammatory agents, immunomodulating agents, growth factors, nucleic acids and antibodies.

18. A method for preparing lipid-based immunogenic artificial particles (1), characterized in that it comprises the steps of: a) preparing a mixture comprising: -an aqueous solution; -lipids;

-glycolipids (5,6) having a lipidic portion (5', 6') and a saccharide portion

(5",6"); and -peptide-lipid conjugates (7,8) having a peptide portion (7', 8') and a lipid portion (7",8"); b) processing said mixture to yield a suspension containing particles (1) comprising: i) a lipid-based matrix (3) having an outer surface (4); ii) at least some of said glycolipids (5,6) embedded into said matrix (3); and iii) at least some of said peptide-lipid conjugates (7,8) embedded into said matrix (3); said particles (1) inducing a cell-mediated immune response against said peptide portion when administered to a host in an immunogenic effective amount.

19. The method of claim 18, characterized in that in said particles(l), the lipidic portion (5',6') of at least some of said glycolipids (5,6) is anchored into said matrix

(3) such that the saccharide portion (5", 6") of said anchored glycolipids (5,6) is projecting at least partially from the outer surface (4) of said matrix (4); and in that the lipidic portion (7', 8') of at least some of said peptide-lipid conjugates (7,8) is anchored into said matrix (3) such that the peptide portion (7", 8") of said anchored peptide-lipid conjugates (7,8) is projecting at least partially from the outer surface

(4) of said matrix (3).

20. The method of claim 18 or 19, characterized in that said processing comprises the step of incubating said suspension at a temperature of about 50°C and the use of an extruder, a fast-vortex or ultrasound in order to shape said suspension into micro-sized particles.

21. A method for inducing a cell-mediated immune response in a host, comprising administering to said host an immunogenic effective amount of artificial immunogenic particles (1), characterized in that said particles comprise: a) a lipid-based matrix (3) having an outer surface (4); b) glycolipids (5,6) embedded into said matrix, said glycolipids (5,6) having a lipidic portion (5', 6') and a saccharide portion (5", 6"); and c) peptide-lipid conjugates (7,8) embedded into said matrix, said peptide-lipid conjugates having a lipidic portion (7', 8') and a peptide portion (7", 8").

22. The method of claim 21 , characterized in that said cell-mediated immune response is directed toward said peptide portion (7", 8").

23. The method of claim 21 or 22, characterized in that in said particles (1), the lipidic portion (5', 6') of at least some of said glycolipids (5,6) is anchored into said matrix (3) such that the saccharide portion (5",6") of said anchored glycolipids (5,6) is projecting at least partially from the outer surface (4) of said matrix (3); and in that the lipidic portion (7', 8') of at least some of said peptide-lipid conjugates (7,8) is anchored into said matrix (3) such that the peptide portion (7", 8") of said anchored peptide-lipid conjugates (7,8) is projecting at least partially from the outer surface (4) of said matrix (3).