WO2006067635A2

WO2006067635A2 - USE OF Ϝδ T LYMPHOCYTE ACTIVATORS AS VACCINE ADJUVANT

Info

Publication number: WO2006067635A2
Application number: PCT/IB2005/004092
Authority: WO
Inventors: Emanuelle Trannoy; Nicolas Burdin
Original assignee: Innate Pharma S.A.
Priority date: 2004-12-20
Filing date: 2005-12-19
Publication date: 2006-06-29
Also published as: WO2006067635A3

Abstract

The present invention is directed to the use of a ϜδT lymphocyte activator, particularly phosphoantigen compounds, as vaccine adjuvant to promote and enhance antigen specific immunological responses, as well as antigen-based vaccines composition comprising a ϜδT lymphocyte activator.

Description

USE OF γδ T LYMPHOCYTE ACTIVATORS AS VACCINE ADJUVANT

FIELD OF THE INVENTION

More specifically, the present invention is directed to the use of a γδT lymphocyte activator, particularly phosphoantigen compounds, as vaccine adjuvant to promote and enhance antigen specific immunological responses, as well as antigen-based vaccines composition comprising a γδT lymphocyte activator.

BACKGROUND OF THE INVENTION

In recent years, a novel lymphoid lineage, γδ T cells, distinct from mainstream T cells, B cells and NK cells, has been identified. Most human adults share a major γδ T cell subset bearing a particular combination of TCR variable regions (Vγ9 and Vδ2), which represents from 60 to 95 % of peripheral blood γδ T cells and up to 1/20 of total T cells in this site (Bottino et al, J Exp Med. 1988 Aug 1 ;168(2):491-505, Davodeau et al, Science. 1993 Jun 18;260(5115): 1800-2). Vγ9Vδ2 T cells and NK cells share several phenotypic features (e.g. frequent expression of so-called Natural Killer receptors) as well as effector properties (NK-like cytolytic activity, release of pro-inflammatory cytokines), which suggest similar physiological roles played by these two lymphoid subsets. In vivo Vγ9Vδ2 T cells are expanded in various physiopathological situations, particularly during (i) infectious processes involving intracellular bacteria (tuberculosis, malaria, tularemia, etc.) and parasites (Ito et al, Chest. 1992; 102(1): 195-7, Modlin et al, Nature. 1989; 339(6225):544- 8), (ii) autoimmune diseases (for a review see Wen and Hayday, Immunol Res. 1997;16(3):229-41. ; and Hayday and Geng, Curr Opin Immunol. 1997; 9(6):884-9. and (iii) several cancers. Besides, Vγ9Vδ2 T cell clones display in vitro both anti-tumor and antibacterial reactivity. In particular they lyse various tumor cell lines (Kobayashi et al, Cancer Immunol Immunother. 2001 ; 50(3): 115-24. These observations suggest an important role for Vγ9Vδ2 T cells in tumor surveillance. Vγ9Vδ2 T cells have also been reported to be involved in the maturation of dendritic cells (DC). Activated γδ T cells induced the production of IL-12 (p40) and IL-12 (p70) by DC, an effect that involved IFN- gamma production, suggesting that gamma delta T cell activation might result in DC maturation and thereby in enhanced alpha beta T cell responses to infection by microorganisms (Ismaili et al, Clin Immunol. 2002; 103(3 Pt 1):296-302).

Vγ9Vδ2 T cells are also activated by small non-peptidic phosphorylated antigens (referred to as "phosphoantigens") that are produced by a wide range of bacteria and intracellular parasites (Constant et al, Science. 1994; 264(5156):267-70). Phosphoantigens are small molecular weight molecules with phosphorylated structures that selectively activate human T cells expressing Vγ9Vδ2 T cells receptors. As described in Constant et al (1994), four molecules named TUBag1-4 were originally isolated from M. tuberculosis, including two antigens that were alkaline phosphatase-sensitive pyrophosphate monoesters of a then unidentified residue, and two larger molecules that were alkaline phosphatase-resistant nucleotidic conjugates containing thymidine-5' triphosphate gamma-diester and uridine-5' triphosphate gamma-diester. The groups of M. Bonneville and JJ. Fournie further characterized the phosphoantigen compounds from mycobacteria and developed pyrophosphate derivatives described (phosphohalohydrins, PHD, described in U.S. Patent No. 6,660,723. A further list of phosphoantigen compounds is described in Espinosa et al, (2001) Microbes and Infection 3: 645-654. The disclosures of each of these references are hereby incorporated by reference, particularly with respect to the phosphoantigen compounds, and methods of making and using them.

The above observations, suggest an involvement of Vγ9Vδ2 T cells in protective immunity against infectious agents. However, to date there have been no effective means for or attempts to apply these observations to the treatment or prevention of disease in vaccine- based approaches.

SUMMARY OF THE INVENTION

The present inventors have discovered that gamma delta T cell-responses induced by the compounds of Formula I may contribute to enhance or augment humoral immune responses to antigens. Accordingly the invention provides a method for inducing a humoral immune response comprising the administration of a composition comprising (a) an antigen and (b) a Vγ9Vδ2 T cell-activating compound to an individual. The invention further concerns the use of an antigen and at least one phosphoantigen compound for the preparation of a medicament for enhancing a humoral immune response of an individual. The Vγ9Vδ2 T cell-activating compound is preferably a phosphoantigen compound, most preferably a compound of Formula 1. Said antigen will generally be a peptide, polypeptide, protein, virus, bacterium, parasite. In a preferred embodiment, the antigen is a toxin-based antigen. Said phosphoantigen compound is preferably provided at a dosage and concentration that provides about 0.10 mg to 1000 mg of compound per kilogram of body weight of said individual, preferably about 0.40 mg to 500 mg of compound per kilogram of body weight of said individual, or more preferably about 0.40 mg to 40 mg of compound per kilogram of body weight of said individual. In another embodiment, the composition preferably comprises at least one phosphoantigen compound at a dosage and concentration that provides at least 0.10 mg, 0.25 mg or 0.4 mg of compound per kilogram of body weight of said individual. Generally, said individual is a mammal, preferably a human.

In preferred aspects of the invention, the phosphoantigen compound is a compound described U.S. Patent No. 6,660,723, or in Espinosa et al, (2001) Microbes and Infection 3: 645-654, particularly a compound selected from the compounds listed in Table 1 , titled "Non-nucleotidic phosphoantigens", of Espinosa et al (2001), or a derivative thereof. In other preferred aspects the phosphoantigen compound is comprises a pyrophosphate moiety. In further preferred aspects, the phosphoantigen compound is a compound of formula (I) :

Formula (I) wherein Cat+ represents at least one identical or different organic or mineral cation(s); m is an integer from 1 to 3;

B is O, NH, or any group capable of being hydrolyzed;

Y = O^"Cat+, a C₁-C₃ alkyl group, a group -A-R₀, or a radical selected from the group consisting of a nucleoside, an oligonucleotide, a nucleic acid, an amino acid, a peptide, a protein, a monosaccharide, an oligosaccharide, a polysaccharide, a fatty acid, a simple lipid, a complex lipid, a folic acid, a tetrahydrofolic acid, a phosphoric acid, an inositol, a vitamin, a co-enzyme, a flavonoid, an aldehyde, an epoxide and a halohydrin; A is O, NH, CHF, CF₂ or CH₂; and,

R₀ and R can be the same or different and are a linear, branched, or cyclic, aromatic, non- aromatic, saturated C₁-C₅₀ Or unsaturated C₁-C₅₀ hydrocarbon group, optionally interrupted by at least one heteroatom, wherein said hydrocarbon group comprises an alkyl, an alkylenyl, or an alkynyl, which can be substituted by one or several substituents selected from the group consisting of: an alkyl, an alkylenyl, an alkynyl, an epoxyalkyl, an aryl, an heterocycle, an alkoxy, an acyl, an alcohol, a carboxylic group (-COOH), an ester, an amine, an amino group (-NH₂), an amide (-CONH₂), an imine, a nitrile, an hydroxyl (-OH), a aldehyde group (-CHO), an halogen, an halogenoalkyl, a thiol (-SH), a thioalkyl, a sulfone, a sulfoxide, and combinations thereof. In further preferred embodiments, the phosphoantigen compound comprises a compound of formula (II):

in which X is a halogen, B is O or NH, m is an integer from 1 to 3, R1 is a methyl or ethyl group, Cat+ represents at least one identical or different organic or mineral cation(s) or protons, n is an integer from 2 to 20, A is O, NH, CHF, CF₂ or CH₂, and Y is O^"Cat+, a nucleoside, or a radical -A-R₀, wherein R₀ is selected from the group of:

1)

OH

— (CH₂)_n — C — R₂

wherein n is an integer from 2 to 20, R₁ is a (CrC₃)alkyl group, and R₂ is an halogenated (Ci-C₃)alkyl, a (C₁-C₃)alkoxy-(C₁-C₃)alkyl, an halogenated (C₂-C₃)acyl or a (C_rC₃)alkoxy- (C₂-C₃JaCyI;

2)

wherein n is an integer from 2 to 20, and R₁ is a methyl or ethyl group; and

3)

R,

Λs

— C — W = C

R₄ \ wherein R₃, R₄, and R₅ , identical or different, are a hydrogen or (CrC₃)alkyl group, W is CH- or -N-, and R₆ is an (C₂-C₃)acyl, an aldehyde, an (C_rC₃)alcohol, or an (C₂-C₃)ester.

In a preferred embodiment, wherein Y is O^"Cat+ or a nucleoside; R1 is a methyl; A is O or CH₂; n is 2; X is a bromide; B is O; and m is 1 or 2. In further aspects, the phosphoantigen compound comprises a compound of formula (III) or (IV) :

in which X is a halogen, m is an integer from 1 to 3, R1 is a methyl or ethyl group, Cat+ represents at least one identical or different organic or mineral cation(s) or protons, n is an integer from 2 to 20, and Y is O^"Cat+, a nucleoside, or a radical -A-R₀, wherein R₀ is selected from the group of:

D

OH

(CH₂J_n C K₂

wherein n is an integer from 2 to 20, R₁ is a (CrC₃)alkyl group, and R₂ is an halogenated (C_rC₃)alkyl, a (CrC^alkoxy-tCrC^alkyl, an halogenated (C₂-C₃)acyl or a (C_rC₃)alkoxy- (C₂-C₃)acyl;

2)

wherein n is an integer from 2 to 20, and R₁ is a methyl or ethyl group; and

3)

R, A

— C — W

R. \ wherein R₃, R₄, and R₅ , identical or different, are a hydrogen or (C_rC₃)alkyl group, W is - CH- or -N-, and Re is an (C₂-C₃)acyl, an aldehyde, an (C_rC₃)alcohol, or an (C₂-C₃)ester.

In further preferred embodiments, the phosphoantigen compound comprises a compound of formula (V):

in which X is an halogen selected from I, Br or Cl, R1 is a methyl or ethyl group, Cat+ represents at least one identical or different organic or mineral cation(s) or protons, and n is an integer from 2 to 20.

In further preferred embodiments, the phosphoantigen compound comprises a compound of formula (Vl):

and x Cat+ is one or two sodium atoms (Na⁺).

In further preferred embodiments, the phosphoantigen compound comprises a compound of formula (VII):

and x Cat+ is one or two sodium atoms (Na⁺).

Preferably, said γδ T cell activator is administered together with a pharmaceutically acceptable carrier. For example, the antigen or combination of antigens and said γδ T cell activator can be administered simultaneously. In a second aspect, the antigen or combination of antigens and said γδ T cell activator are administered sequentially. It will be appreciated that the γδ T cell activator can be administered prior to, concurrently with or subsequent to administration of an antigen or a combination of antigens to a subject for immunization purposes. Preferably, said antigen or combination of antigens are microbial antigens, preferably, viral, bacterial, fungal, protozoan, yeast or parasite antigens. In a preferred embodiment, said antigen is a recombinant polypeptide antigen, preferably a purified or substantially pure polypeptide composition. Optionally, said antigen or combination of antigens is a tumoral antigen, a viral or bacterial antigen, or a toxin-based antigen.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the antigen specific antibody responses raised in immunized animals, as tested by standards ELISAs and expressed as ELISA titers (Iog10). Antibody titers obtained after 2 HIV Tat (10μg/ml of Tat) injections (W17) were compared to pre-immune sera (WO and W10) of the corresponding animals. When administered with BrHPP, the Tat immunisation elicited a greater Tat specific antibody response over that observed with Tat alone.

DETAILED DESCRIPTION OF THE INVENTION

Within the meaning of the present invention, the term "conjoint administration" is used to refer to administration of an immune adjuvant and an antigen simultaneously in one composition, or simultaneously in different compositions, or sequentially. For the sequential administration to be considered "conjoint", however, the antigen and adjuvant must be administered separated by a time interval that still permits the adjuvant to augment the immune response to the antigen. For example, when the antigen is a polypeptide, the antigen and adjuvant are administered on the same day (e.g. within 24 hours of one another), preferably within an hour of each other, and most preferably simultaneously. However, when nucleic acid is delivered to the subject and the polypeptide antigen is expressed in the subject's cells, the adjuvant is administered within 24 hours of nucleic acid administration, preferably within 6 hours.

As used herein, the term "immunogenic" means that an agent is capable of eliciting a humoral or cellular immune response, and preferably both. An immunogenic entity is also antigenic. An immunogenic composition is a composition that elicits a humoral or cellular immune response, or both, when administered to an animal having an immune system.

The term "antigen" refers to any agent (e.g., protein, peptide, lipid, polysaccharide, glycoprotein, glycolipid, nucleic acid or any combination of any of the foregoing) that, when introduced into a host, animal or human, having an immune system (directly or upon expression as in, e.g., DNA vaccines), is recognized by the immune system of the host and is capable of eliciting an immune response. As defined herein, the antigen-induced immune response can be humoral or cell-mediated, or both. An agent is termed "antigenic" when it is capable of or comprises a component capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor (TCR). Antigens may include but are not limited to "surface antigens", i.e., expressed naturally on the surface of a pathogen, or the surface of an infected cell, or the surface of a tumor cell. A molecule that is antigenic need not be itself immunogenic, i.e., capable of eliciting an immune response without an adjuvant or carrier. An antigen may be "species-specific", referring to an antigen that is only present in or derived from a particular species.

A "vaccine" is an immunogenic or antigenic composition that can be used to elicit protective immunity or a protective immune response in a recipient. The protective immunity may be towards pathogens such as viruses, fungi, parasites, yeast, bacteria, and protozoa. More particularly, said composition is capable of eliciting protection against infections, whether partial or complete. A vaccine may also be useful for treatment of an individual, in which case it is called a therapeutic vaccine. Said vaccine compositions may include prophylactic as well as therapeutic vaccine compositions. The term "DNA vaccine" is an informal term of art, and is used herein to refer to a vaccine delivered by means of a recombinant vector. An alternative, and more descriptive term used herein is "vector vaccine" (since some potential vectors, such as retroviruses and Antiviruses are RNA viruses, and since in some instances non-viral RNA instead of DNA is delivered to cells through the vector). Generally, the vector is administered in vivo, but ex vivo transduction of appropriate antigen presenting cells, such as dendritic cells (DC), with administration of the transduced cells in vivo, is also contemplated. The term "treat" is used herein to mean to relieve or alleviate at least one symptom of a disease in a subject. Within the meaning of the present invention, the term "treat" may also mean to prolong the prepatency, i.e., the period between infection and clinical manifestation of a disease. Preferably, the disease is either infectious disease (e.g., viral, bacterial, parasitic, or fungal) or malignancy (e.g., solid or blood tumors such as sarcomas, carcinomas, gliomas, blastomas, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma, leukemia, melanoma, etc.).

The term "protect" is used herein to mean prevent or treat, or both, as appropriate, development or continuance of a disease in a subject. Within the meaning of the present invention, the disease is selected from the group consisting of infection (e.g., viral, bacterial, parasitic, or fungal) and malignancy (e.g., solid or blood tumors such as sarcomas, carcinomas, gliomas, blastomas, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma, leukemia, melanoma, etc.). For example, a therapeutic administration of a tumor-specific antigen conjointly with an adjuvant comprising a compound of Formula I can enhance an anti-tumor immune response leading to slow-down in tumor growth and metastasis or even tumor regression.

The term "protective immunity" refers to an immune response in a host animal (either active/acquired or passive/innate, or both) which leads to inactivation and/or reduction in the load of said antigen and to generation of long-lasting immunity (that is acquired, e.g., through production of antibodies), which prevents or delays the development of a disease upon repeated exposure to the same or a related antigen. A "protective immune response" comprises a humoral (antibody) immunity or cellular immunity, or both, effective to, e.g., eliminate or reduce the load of a pathogen or infected cell (or produce any other measurable alleviation of the infection), or to reduce a tumor burden in an immunized (vaccinated) subject. Within the meaning of the present invention, protective immunity may be partial.

As used herein, the term "augment the immune response" means enhancing or extending the duration of the immune response, or both. When referred to a property of an agent (e.g., adjuvant), the term "able to augment the immunogenicity" refers to the ability to enhance the immunogenicity of an antigen or the ability to extend the duration of the immune response to an antigen, or both.

The phrase "enhance immune response" within the meaning of the present invention refers to the property or process of increasing the scale and/or efficiency of immunoreactivity to a given antigen, said immunoreactivity being either humoral or cellular immunity, or both. An immune response is believed to be enhanced, if any measurable parameter of antigen-specific immunoreactivity (e.g., antibody titer, T cell production) is increased at least two-fold, preferably ten-fold, most preferably thirty-fold.

The term "therapeutically effective" applied to dose or amount refers to that quantity of a compound or pharmaceutical composition or vaccine that is sufficient to result in a desired activity upon administration to a mammal in need thereof. As used herein with respect to adjuvant — and antigen-containing compositions or vaccines, the term "therapeutically effective amount/dose" is used interchangeably with the term "immunogenically effective amount/dose" and refers to the amount/dose of a compound (e.g., an antigen and/or an adjuvant of the invention) or pharmaceutical composition or vaccine that is sufficient to produce an effective immune response upon administration to a mammal.

The phrase "pharmaceutically acceptable", as used in connection with compositions of the invention, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

The term "delivery system" applied to pharmaceutical or vaccine compositions of the invention refers to a diluent, excipient, or vehicle with which a compound (e.g., an antigen and/or an adjuvant comprising glycosylceramide) is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution, saline solutions, and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 18^th Edition.

The term "native antibodies" or "immunoglobulins" refers to usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Clothia et al., J MoI. Biol., 186: 651-663, 1985; Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82: 4592-4596, 1985). The term "antibody" or "Ab" is used in the broadest sense and specifically covers not only native antibodies but also single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, as well as antibody fragments (e.g., Fab, F(ab')2, scFv and Fv), so long as they exhibit the desired biological activity.

"Cytokine" is a generic term for a group of proteins released by one cell population which act on another cell population as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are interferons (IFN, notably IFN-γ), interleukins (IL, notably IL-1 , IL-2, IL-4, IL- 10, IL-12), colony stimulating factors (CSF), thrombopoietin (TPO), erythropoietin (EPO), leukemia inhibitory factor (LIF), kit-ligand, growth hormones (GH), insulin-like growth factors (IGF), parathyroid hormone, thyroxine, insulin, relaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), hematopoietic growth factor, hepatic growth factor, fibroblast growth factors (FGF), prolactin, placental lactogen, tumor necrosis factors (TNF), mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor (VEGF), integrin, nerve growth factors (NGF), platelet growth factor, transforming growth factors (TGF), osteoinductive factors, etc.

The term "subject" as used herein refers to an animal having an immune system, preferably a mammal or more preferably a primate. Preferably the term refers to humans.

The term "about" or "approximately" usually means within 20%, more preferably within 10%, and most preferably still within 5% of a given value or range. Alternatively, especially in biological systems (e.g., when measuring an immune response), the term "about" means within about a log (i.e., an order of magnitude) preferably within a factor of two of a given value.

The terms "vector", "cloning vector", and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and/or translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are well-known and are explained fully in the literature. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989)

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and Il (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

A "nucleic acid molecule" (or alternatively "nucleic acid") refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine: "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine: "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Oligonucleotides (having fewer than 100 nucleotide constituent units) or polynucleotides are included within the defined term as well as double stranded DNA- DNA, DNA-RNA, and RNA-RNA helices. This term, for instance, includes double-stranded DNA found, inter alia, in linear (e.g., restriction fragments) or circular DNA molecules, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation.

As used herein, the term "polypeptide" refers to an amino acid-based polymer, which can be encoded by a nucleic acid or prepared synthetically. Polypeptides can be proteins, protein fragments, chimeric proteins, etc. Generally, the term "protein" refers to a polypeptide expressed endogenously in a cell. Generally, a DNA sequence encoding a particular protein or enzyme is "transcribed" into a corresponding sequence of mRNA. The mRNA sequence is, in turn, "translated" into the sequence of amino acids which form a protein. An "amino acid sequence" is any chain of two or more amino acids. The term "peptide" is usually used for amino acid-based polymers having fewer than 100 amino acid constituent units, whereas the term "polypeptide" is reserved for polymers having at least 100 such units. Herein, however, "polypeptide" will be the generic term. As used herein, an immunogenic composition elicits an immune response in a mammal to whom the composition is administered. The compounds according to the invention preferably elicit a humoral-mediated response. As used herein, a vaccine composition is a composition which elicits an immune response in a mammal to whom the composition is administered and which protects the mammal from subsequent challenge or infection with the antigen of the composition or with a related organism. As used herein, "protection", for example protection against a virus, refers to generation of an immune response in the mammal (e.g., primate) which is protective (partially or totally) against manifestations of the disease caused by the antigen in the composition or a related organism, e.g. the virus. A vertebrate that is protected against disease caused by a virus may be infected with the virus, but to a lesser degree than would occur without immunization; may be infected with the virus, but does not exhibit disease symptoms; or may be infected with virus, but exhibits fewer disease symptoms than would occur without immunization. Alternatively, the vertebrate that is protected against disease caused by virus may not become infected with the virus at all, despite exposure to the virus.

Adjuvant composition, Vaccine composition, and uses thereof

The present invention provides a vaccine or adjuvant composition comprising a γδ T cell activating compound, preferably a Vγ9Vδ2 T cell activating compound. Preferably the γδ T cell activator or Vγ9Vδ2 T cell activator is a phosphoantigen compound, preferably a compound of Formula I, preferably a compound of Formula Il to VII, more preferably BrHpp, and optionally a pharmaceutical acceptable carrier, diluent or excipient.

Examples of pharmaceutically acceptable delivery system that can be used in accordance with the invention include typically large, slowly metabolizing macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Examples of suitable carriers that also act as stabilizers for peptides include, without limitation, pharmaceutical grades of dextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran, and the like. Other suitable carriers include, again without limitation, starch, cellulose, sodium or calcium phosphates, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycols (PEGs), and combination thereof. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in REMINGTONS PHARMACEUTICAL SCIENCES (Mack Pub. Co. , N. J. 1991), incorporated herein by reference. In a preferred embodiment, the pharmaceutical carrier is in the form of a lipid dispersion, more preferably the lipid dispersion consists of liposomes. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol.

The present invention encompasses the use of a composition comprising a γδT cell activator, preferably wherein said activator is a phosphoantigen compound, preferably a compound of Formula I, preferably a compound of Formula Il to VII, more preferably BrHpp, as a vaccine adjuvant.

More particularly, the invention relates to a vaccine composition comprising a therapeutically effective amount of a vaccine and an amount of a γδT cell activator, preferably wherein said activator is a phosphoantigen compound, preferably a compound of Formula I, preferably a compound of Formula Il to VII, more preferably BrHpp sufficient to augment an immune response or preferably to enhance an immune response. The invention also concerns the use of a vaccine composition comprising a therapeutically effective amount of a vaccine and an amount of a γδT cell activator for the preparation of a composition for augmenting an immune response or preferably for enhancing an immune response

The adjuvant of the invention can be administered as part of a pharmaceutical or vaccine composition comprising an antigen or as a separate formulation, which is administered conjointly with a second composition containing an antigen. In any of these compositions γδ T cell activator can be combined with other adjuvants and/or excipients/carriers. These other adjuvants include, but are not limited to, oil-emulsion and emulsifier-based adjuvants such as complete Freund's adjuvant, incomplete Freund's adjuvant, MF59, or SAF; mineral gels such as aluminum hydroxide (alum), aluminum phosphate or calcium phosphate; microbially-derived adjuvants such as cholera toxin (CT), pertussis toxin, Escherichia coli heat-labile toxin (LT), mutant toxins (e.g., LTK63 or LTR72), Bacille Calmette-Guerin (BCG), Corynebacterium parvum, DNA CpG motifs, muramyl dipeptide, or monophosphoryl lipid A; particulate adjuvants such as immunostimulatory complexes (ISCOMs), liposomes, biodegradable microspheres, or saponins (e.g., QS-21); synthetic adjuvants such as nonionic block copolymers, muramyl peptide analogues (e.g., N-acetyl- muramyl-L-threonyl-D-isoglutamine [thr-MDP], N-acetyl-nor-muramyl-L-alanyl-D- isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine^-fi'^'-dipalmitoyl-sn- glycero-3-hydroxyphosphoryloxy]-ethylamine), polyphosphazenes, or synthetic polynucleotides, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, hydrocarbon emulsions, or keyhole limpet hemocyanins (KLH). Other preferred adjuvants include cytokines. Certain cytokines, for example TRANCE, flt- 3L, and CD40L, enhance the immunostimulatory capacity of APCs. Non-limiting examples of cytokines which may be used alone or in combination include, interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 α), interleukin-11 (IL-11), MIP-Ia, leukemia inhibitory factor (LIP), c-kit. ligand, thrombopoietin (TPO), CD40 ligand (CD40L), tumor necrosis factor-related activation-induced cytokine (TRANCE) and flt3 ligand (flt-3L). Cytokines are commercially available from several vendors such as, for example, Genzyme, Genentech, Amgen and Immunex. Preferably, these additional adjuvants are also pharmaceutically acceptable for use in humans.

An adjuvant compound according to the invention may be administered with a priming dose of antigen. An adjuvant compound according to the invention may then be administered again with the boost dose. Alternatively, an adjuvant compound according to the invention is administered with a boost dose of antigen. In another aspect an adjuvant compound of according to the invention is administered with a boost dose of antigen but not with the priming dose of antigen. A "prime dose" is the first dose of antigen administered to the subject. In the case of a subject that has an infection the prime dose may be the initial exposure of the subject to the infectious microbe and thus the adjuvant is administered to the subject with the boost dose. A "boost dose" is a second or third, etc, dose of antigen administered to a subject that has already been exposed to the antigen. In some cases the prime dose administered with the adjuvant is so effective that a boost dose is not required to protect a subject at risk of infection from being infected. As further described herein in the section titled Examples, the inventors note that in some embodiments it may be preferable to administer the adjuvant with the first dose of a particular antigen(s) administered to the subject, e.g. preferably the adjuvant of the invention is administered conjointly with the prime dose of antigen. In this embodiment, the adjuvant of the invention may optionally be administered additionally conjointly with one or more subsequent boost doses of antigen.

The vaccine composition is preferably a pharmaceutical composition comprising one or several antigens (or antigenic molecules), for combined, separate or sequential use. Antigens, as further described throughout the present disclosure, can be for example in the form of a synthetic or natural peptide, a lipid, a recombinant polypeptide, a nucleic acid, a killed, inactivated or attenuated pathogen, microorganism, or parasite. A vaccine can be for example an antigen such as protein or peptide in enriched or purified form, or a antigen delivered by a vector such as for example an adenoviral vector, Sindbis virus vector, or pox virus vector. An antigen can be entire protein or any epitope-containing fragment or portion thereof, particularly peptides that are presented to the immune system through MHC class I or MHC class Il molecules. Examples include any viral antigen, bacterial antigen, parasite antigen, tumor antigen, etc.

There are at least six major types of vaccines: inactivated vaccines, live attenuated vaccines, toxoids, antigenic extracts (purified or partially purified antigen), purified recombinant proteins, synthetic vaccines, and genetically engineered vaccines. Thus, a mammalian host may be vaccinated (immunized) against polio by the use of either an inactivated or attenuated vaccine, whereas vaccination (immunization) against tetanus is via the use of tetanus toxoid. Vaccination against a microbial infection may be by the use of an inactivated agent (e.g., killed Bordetella pertussis in the case of pertussis vaccine), attenuated agent (e.g., BCG in the case of tuberculosis vaccine), or purified antigen (e.g., purified polysaccharide from Neisseria meninaitidis in the case of meningitis vaccine). The prevention of the growth or establishment of a tumor may be accomplished through the use of a purified or partially purified antigen vaccine, or by the use of the tumor cells themselves (in a protocol analogous to the use of attenuated or killed microbial vaccines).

A vaccine composition as described by the invention can be used to immunize humans or animals against different diseases (adjuvant). It is contemplated that the organism, host, or subject to which the compositions will be administered will be an animal or human.

The subject to whom the composition is administered may be naive or alternatively currently infected or diseased, or recovered from a past infection or disease state. The composition may be administered more than once, and in a specific embodiment at least twice or thrice. The administration of the vaccine composition can be a priming or a boosting administration.

The compositions can be administered to humans and animals either orally, rectally, parenterally (intravenous, by intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops), or as a buccal or nasal spray.

The present invention is directed to a method of improving vaccine potency in a mammalian host comprising administering a therapeutically effective amount of a phosphoantigen compound, preferably a compound of Formula I₁ preferably a compound of Formula Il to VII, more preferably BrHpp. Preferably, said γδT cell activator is as a pharmaceutical composition comprising said γδT cell activator and a pharmaceutically acceptable carrier. The present invention also concerns the use of a phosphoantigen compound for the preparation of a composition for improving vaccine potency in a mammalian host. The composition can further comprises a vaccine.

More particularly, the present invention is directed to a method of improving the potency of a vaccine in a subject comprising the steps of:

(a) administering to said subject a therapeutically effective amount of a vaccine; and, (b) administering to said subject an immune response enhancing amount of a

Vγ9Vδ2 T cell activator compound according to the invention.

Preferably, the vaccine composition comprising one or several antigens and the γδ T cell activator are administered simultaneously. Alternatively, the vaccine composition and the γδ T cell activator are administered sequentially. Preferably the antigen-containing composition and the γδ T cell activator are administered within 48 hours of one another, preferably within 24 hours, 12 hours or 6 hours of one another.

The present invention relates to the use of a γδ T cell activator for the preparation of a medicament for the treatment and/or prevention of a disease whereby stimulation of a humoral response is needed. Preferably, said disease is an infection. In particularly preferred embodiments, the infection Indeed, the γδ T cell activators are used as an adjuvant to elicit a B cell response in vivo.

Another object of the invention relates to the use of a γδ T cell activator described herein, for the manufacture of a pharmaceutical composition to enhance or augment an immune response.

A further particular object of the present invention concerns a method of causing or enhancing or augmenting an antigen-specific immune response in a subject, comprising administering to a subject said antigen and a composition comprising a γδ T cell activator of Formula I to VII. The composition may be administered simultaneously, a few days before or sequentially with said antigen in order to obtain and/or stimulate an antigen- specific immune response in a subject. Toxin-based vaccines

In a preferred embodiment, the invention can be used to augment an immune response in an individual against a toxin, preferably a bacterial toxin. Since the vaccine adjuvants of the invention are capable of enhancing a humoral immune response, this may be particularly well suited to use with vaccines against toxins. Accordingly, it is an object of the present invention to provide novel vaccines comprising a toxin or toxin-derived composition and an adjuvant composition comprising a γδ T cell activator. The antigen is generally a mutant or de-toxified toxin derivative or antigenic fragments thereof, which, when administered to animals or humans conjointly with the adjuvant of the invention, are capable of inducing production of protective antibodies directed against a particular toxin and thereby providing prophylaxis against infection by the disease states resulting from such infection, and/or from the toxin itself. It is a particular aim of the present invention to provide enhanced vaccine compositions that are safe and have increased ability to stimulate a humoral response against the pathogen. In such embodiments, the compounds of the invention are preferably capable of augmenting a humoral immune response observed with the antigen over that observed with the antigen in the absence of the adjuvant. For example, in a preferred aspect the method comprises eliciting a humoral response against a toxin from Bacillus or Clostridium, preferably against a toxin produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme, C. diphtheriae.

The toxin is preferably a enriched, isolated or purified toxin or toxin derivative. In further aspects recombinant nucleic acids encoding the mutants, or antigenic fragments thereof, plasmids comprising such nucleic acids and cell lines comprising these plasmids or recombinant DNA itself is administered, such that expression of the toxin or mutant toxin may be achieved. Such recombinant DNA can be provided by PCR amplification of the DNA encoding for the desired sequence. Most preferred are purified derivatives (mutants) of a toxin deficient in at least one aspect of the toxin's cytolytic mechanism.

Examples include among others cholera toxin, tetanus toxin, botulism toxin, the plant- derived ricin toxin (ricin A chain), staphylococcal enterotoxin b, and trichothecene mycotoxins. In one example, International Patent Publication no. WO 03/005957 describes the use of a mutant C. septicum alpha toxin defective in at least one of three aspects of the native toxin's cytolytic mechanism, including (1) receptor binding, (2) membrane insertion and pore formation, and (3) cellular activation, described are purified forms of these alpha toxin mutants or antigenic fragments thereof, which have sufficiently diminished hemolytic activity and diminished receptor binding can be used as vaccines against C. septicum disease since alpha toxin is the only lethal factor produced by C. septicum and immunity to this toxin likely plays a major role in immunity to disease caused by C. septicum. in other examples, a mutant cholera toxin a subunit (MCT-A) and heat labile toxin B subunit (LT-B) is described. (Yuki and Kiyono, Rev Med Virol. 2003; 13(5): 293-310). other examples include a polypeptide that represents a portion (in this case a third) of the native toxin molecule retains the ability to be adsorbed from the airway and to evoke an immune response but retains none of the adverse effects of the native toxin, such that this polypeptide can serve as a carrier molecule in the creation of inhalation vaccines against a pathogen (Park and Simpson, Expert Rev. Vaccines. 2004;3(4):477- 87). further examples include anthrax vaccines, for example monovalent and bivalent anthrax plasmid DNA (pDNA) vaccines encoding genetically detoxified protective antigen (PA) and lethal factor (LF) proteins were able to protect rabbits from an aerosolized inhalation spore challenge. (Hermanson et al. Proc. Natl. Acad. Sci. USA. 2004; 101 (37): 13601 -6. the disclosures of each of the above-listed references are incorporated herein by reference.

Microbial infections Preferably, said compositions of the invention prevent a microbial infection. Said microbial infection is caused by a microbe selected from the group consisting of viruses, fungi, yeast, bacteria, and protozoa.

A "microbial antigen" as used herein is an antigen of a microorganism and includes but is not limited to infectious virus, infectious bacteria, infectious parasites and infectious fungi. Such antigens include the intact microorganism as well as natural isolates and fragments or derivatives thereof and also synthetic compounds which are identical to or similar to natural microorganism antigens and induce an immune response specific for that microorganism. A compound is similar to a natural microorganism antigen if it induces an immune response (humoral and/or cellular) to a natural microorganism antigen. Most such antigens are used routinely in the art and are well known to those of ordinary skill in the art. Another example is a peptide mimic of a polysaccharide antigen.

Vaccines and antigens may be derived from infectious virus of both human and non- human vertebrates, include retroviruses, RNA viruses and DNA viruses. This group of retroviruses includes both simple retroviruses and complex retroviruses. The simple retroviruses include the subgroups of B-type retroviruses, C-type retroviruses and D-type retroviruses. An example of a B-type retrovirus is mouse mammary tumor virus (MMTV). The C-type retroviruses include subgroups C-type group A (including Rous sarcoma virus (RSV), avian leukemia virus (ALV), and avian myeloblastosis virus (AMV)) and C-type group B (including murine leukemia virus (MLV), feline leukemia virus (FeLV), murine sarcoma virus (MSV), gibbon ape leukemia virus (GALV), spleen necrosis virus (SNV), reticuloendotheliosis virus (RV) and simian sarcoma virus (SSV)). The D-type retroviruses include Mason-Pfizer monkey virus (MPMV) and simian retrovirus type 1 (SRV-1). The complex retroviruses include the subgroups of lentiviruses, T-cell leukemia viruses and the foamy viruses. Lentiviruses include HIV-1 , but also include HIV-2, SIV, Visna virus, feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV). The T-cell leukemia viruses include HTLV-1 , HTLV-II, simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV). The foamy viruses include human foamy virus (HFV), simian foamy virus (SFV) and bovine foamy virus (BFV).

Examples of other RNA viruses that are antigens in mammals include, but are not limited to, the following: members of the family Reoviridae, including the genus Orthoreovirus (multiple serotypes of both mammalian and avian retroviruses), the genus Orbivirus (Bluetongue virus, Eugenangee virus, Kemerovo virus, African horse sickness virus, and Colorado Tick Fever virus), the genus Rotavirus (human rotavirus, Nebraska calf diarrhea virus, murine rotavirus, simian rotavirus, bovine or ovine rotavirus, avian rotavirus); the family Picornaviridae, including the genus Enterovirus (poliovirus, Coxsackie virus A and B, enteric cytopathic human orphan (ECHO) viruses, hepatitis A virus, Simian enteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirus muris, Bovine enteroviruses, Porcine enteroviruses , the genus Cardiovirus (Encephalomyocarditis virus (EMC), Mengovirus), the genus Rhinovirus (Human rhinoviruses including at least 113 subtypes; other rhinoviruses), the genus Apthovirus (Foot and Mouth disease (FMDV); the family Calciviridae, including Vesicular exanthema of swine virus, San Miguel sea lion virus, Feline picornavirus and Norwalk virus; the family Togaviridae, including the genus Alphavirus (Eastern equine encephalitis virus, Semliki forest virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus), the genus Flavirius (Mosquito borne yellow fever virus, Dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, West Nile virus, Kunjin virus, Central European tick borne virus, Far Eastern tick borne virus, Kyasanur forest virus, Louping III virus, Powassan virus, Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosal disease virus, Hog cholera virus, Border disease virus); the family Bunyaviridae, including the genus Bunyvirus (Bunyamwera and related viruses, California encephalitis group viruses), the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever virus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus,

Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi and related viruses); the family Orthomyxoviridae, including the genus Influenza virus (Influenza virus type A, many human subtypes); Swine influenza virus, and Avian and Equine Influenza viruses; influenza type B (many human subtypes), and influenza type C (possible separate genus); the family paramyxoviridae, including the genus Paramyxovirus (Parainfluenza virus type 1 , Sendai virus, Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measles virus, subacute sclerosing panencephalitis virus, distemper virus, Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice); forest virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus), the genus Flavirius (Mosquito borne yellow fever virus, Dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, West Nile virus, Kunjin virus, Central European tick borne virus, Far Eastern tick borne virus, Kyasanur forest virus, Louping III virus, Powassan virus, Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosal disease virus, Hog cholera virus, Border disease virus); the family Bunyaviridae, including the genus Bunyvirus (Bunyamwera and related viruses, California encephalitis group viruses), the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever virus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi and related viruses); the family Orthomyxoviridae, including the genus Influenza virus (Influenza virus type A, many human subtypes); Swine influenza virus, and Avian and Equine Influenza viruses; influenza type B (many human subtypes), and influenza type C (possible separate genus); the family paramyxoviridae, including the genus Paramyxovirus (Parainfluenza virus type 1 , Sendai virus, Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measles virus, subacute sclerosing panencephalitis virus, distemper virus, Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice); the family Rhabdoviridae, including the genus Vesiculovirus (VSV), Chandipura virus, Flanders-Hart Park virus), the genus Lyssavirus (Rabies virus), fish Rhabdoviruses, and two probable Rhabdoviruses (Marburg virus and Ebola virus); the family Arenaviridae, including Lymphocytic choriomeningitis virus (LCM), Tacaribe virus complex, and Lassa virus; the family Coronoaviridae, including Infectious Bronchitis Virus (IBV), Mouse Hepatitis virus, Human enteric corona virus, and Feline infectious peritonitis (Feline coronavirus). Illustrative DNA viruses that are antigens in mammals include, but are not limited to: the family Poxviridae, including the genus Orthopoxvirus (Variola major, Variola minor, Monkey pox Vaccinia, Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus (Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avian poxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genus Suipoxvirus

(Swinepox), the genus Parapoxvirus (contagious postular dermatitis virus, pseudocowpox, bovine papular stomatitis virus); the family Iridoviridae (African swine fever virus, Frog viruses 2 and 3, Lymphocystis virus of fish); the family Herpesviridae, including the alpha- Herpesviruses (Herpes Simplex Types 1 and 2, Varicella-Zoster, Equine abortion virus, Equine herpes virus 2 and 3, pseudorabies virus, infectious bovine keratoconjunctivitis virus, infectious bovine rhinotracheitis virus, feline rhinotracheitis virus, infectious laryngotracheitis virus) the Beta-herpesvirises (Human cytomegalovirus and cytomegaloviruses of swine, monkeys and rodents); the gamma-herpesviruses (Epstein- Barr virus (EBV), Marek's disease virus, Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus, guinea pig herpes virus, Lucke tumor virus); the family Adenoviridae, including the genus Mastadenovirus (Human subgroups A₁B₁C₁D₁E and ungrouped; simian adenoviruses (at least 23 serotypes), infectious canine hepatitis, and adenoviruses of cattle, pigs, sheep, frogs and many other species, the genus Aviadenovirus (Avian adenoviruses); and non-cultivatable adenoviruses; the family Papoviridae, including the genus Papillomavirus (Human papilloma viruses, bovine papilloma viruses, Shope rabbit papilloma virus, and various pathogenic papilloma viruses of other species), the genus Polyomavirus (polyomavirus, Simian vacuolating agent (SV-40), Rabbit vacuolating agent (RKV), K virus, BK virus, JC virus, and other primate polyoma viruses such as Lymphotrophic papilloma virus); the family Parvoviridae including the genus Adeno- associated viruses, the genus Parvovirus (Feline panleukopenia virus, bovine parvovirus, canine parvovirus, Aleutian mink disease virus, etc). Finally, DNA viruses may include viruses which do not fit into the above families such as Kuru and Creutzfeldt-Jacob disease viruses and chronic infectious neuropathic agents (CHINA virus).

Specific examples of HIV antigen can be, without any limitation, one or several antigens derived from a product selected from the group consisting of Tat, gp120, gp160, gag, pol, protease, and nef. Preferably, the HIV antigen is an antigen derived from gp120.

Other preferred exemplary antigens are HPV antigens from any strain of HPV. HPV expresses six or seven non-structural and two structural proteins. Viral capsid proteins L1 and L2 are the late structural proteins. L1 is the major capsid protein, the amino acid sequence of which is highly conserved among different HPV types. There are seven early non-structural proteins. Proteins E1 , E2, and E4 play an important role in virus replication.

Protein E4 also plays a role in virus maturation. The role of E5 is less well known. Proteins E6 and E7 are oncoproteins critical for viral replication, as well as for host cell immortalization and transformation. Fusion proteins of the invention can contain either the entire sequence of an HPV protein or a fragment thereof, e.g., a fragment of at least 8 amino acids. In one embodiment, the HPV antigenic sequence is derived from a "high risk" HPV, such as HPV16 or HPV18 E7 protein. The HPV antigenic sequence can include an MHC-binding epitope, e.g., an MHC class I and/or an MHC class Il binding epitope.

Further preferred exemplary antigens are those obtained or derived from the hepatitis family of viruses, including hepatitis A virus (HAV), hepatitis B virus (BBV), hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis E virus (BEV) and hepatitis G virus (HGV). See, e. g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. The HCV genome encodes several viral proteins, including E I and E2. See, e. g., Houghton et al. (1991) Hepatology 14: 381-388. Nucleic acid molecules containing sequences encoding these proteins, as well as antigenic fragments thereof, will find use in the present methods. Similarly, the coding sequence for the 8-antigen from HDV is known (see U. S. Patent No. 5,378,814). In like manner, a wide variety of proteins from the herpesvirus family can be used as antigens in the present invention, including proteins derived from herpes simplex virus (HSV) types 1 and 2, such as HSV- 1 and HSV-2 glycoproteins gB, gD and gH; antigens from varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) including CMV gB, and gH; and antigens fi-om other human heipesviruses such as HHV6 and HAV7. (See, e. g. Chee et al. (1990) Cytomegaloviruses (J. K. McDougall, ed., Springer Verlag, pp. 125-169; McGeoch et al. (1988) J. Gen. Virol. 69: 1531-1574; U. S. Patent No. 5,171 ,568: Baer et al. (1984) Nature 310: 207-21 1 ; and Davison et al. (1986) J. Gen. Virol. 67: 1759 )

Antigens or vaccines may be derived from respiratory syncytial virus (RSV), a negative strand virus of theparamyxoviridae family and a major cause of lower pulmonary tract disease, particularly in young children and infants. RSV contains two prominent outer envelope glycoproteins, fusion (F) protein and attachment (G) protein, that are important for viral infectivity and thus serve as reasonable targets for the design of a subunit vaccine to RSV. The wild type (native) nucleotide and amino acid sequences of the RSV F protein are known in the art (Collins et al_, Proc. Natl. Acad. Sci (USA) 81 :7683-7687 (1984); U.S. Patent No. 5,639,853; U.S. Patent No. 5,723,130). Preferred RSV proteins suitable for use in the invention include the complete RSV F protein as well as functional portions of the RSV F protein. For example, a functional portion can be a portion of the protein which retains the ability to induce an antibody response when administered to a mammal. Examples of such immunogenic portions are polypeptides comprising amino acid positions 283-315, 289-315 and 294-299 of the RSV F protein. These regions include an epitope of the RSV F protein which elicits both neutralizing and antifusion antibodies (U.S. Patent 5,639,853). Alternatively, an RSV F protein in its native dimeric form (140 kD) may be used (U.S. Patent 5,223,254).

Other exemplary vaccines used in accordance with the adjuvants of the invention include Influenza Virus Vaccines. Trivalent A & B Live, Cold Adapted or FluMist (Medimmune Vaccines) is an aqueous nasal spray trivalent formulation of natural recombinant (reassortment; not gene spliced) cold-adapted temperature-sensitive attenuated nonpathogenic live influenza viruses having immunogenic viral coat proteins (hemagglutinin and neuraminidase) from representative virulent epidemic wild-type influenza strains and an influenza virus core with six attenuating gene mutations.

Preferred recombinant cold-adapted/temperature-sensitive influenza virus strains that can be used as vaccines have a viral coat presenting influenza virus hemagglutinin (HA) and neuraminidase (NA) immunogenic epitopes from a virulent influenza strain along with an attenuated influenza virus core. The HA and NA RNA sequences of an attenuated master donor virus (MDV) are replaced with HA and NA RNA sequences from epidemic wild-type influenza strains. Temperature sensitivity is conferred to the MDV by modification of the polymerase Basic Protein 2 (PB2) gene, which encodes a 759 amino acid polypeptide that is one of the three proteins comprising the RNA-dependent polymerase complex of influenza virus. Viral RNA replication is dependent on PB2 (along with PB1 , PA, and NP). The three polymerase proteins, PB1 , PB2, and PA, form a trimolecular complex in the nuclei of infected cells

Other vaccines and antigens may be derived from bacteria, parasites or yeast. Examples of suitable species include Neisseria spp, including N. gonorrhea and N. meningitidis (for example, capsular polysaccharides and conjugates thereof, transferrin-binding proteins, lactoferrin binding proteins, PiIC and adhesions can be used as antigens); S. pyogenes (for example M proteins or fragments thereof, C5A protease, lipoteichoic acids), S. agalactiae, S. mutans; H. ducreyi; Moraxella spp, including M. catarrhalis, also known as Branhamella catarrhalis (for example high and low molecular weight adhesins and invasins); Bordetella spp, including B. pertussis (for example pertactin, pertussis toxin or derivatives thereof, filamenteous hemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B. bronchiseptica; Mycobacterium spp., including M. tuberculosis (for example ESAT6, Antigen 85A, -B or -Q, M. bovis, M leprae, M avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coli (for example colonization factors, heat-labile toxin or derivatives thereof, heat-stable toxin or derivatives thereof), enterohemorragic E. coli, enteropathogenic E. coli (for example io Shiga toxin-like toxin or derivatives thereof); Vibrio spp, including V. cholera (for example cholera toxin or derivatives thereop; Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y enterocolitica (for example a Yop protein) , Y. pestis, Y. pseudotuberculosis; Campylobacter spp, including C jejuni (for example toxins, adhesins and invasins) and C coli; Salmonella spp, including S. typhip S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp., including L. monocytogenes; Helicobacter spp, including H. pylori (for example urease, catalase, vacuolating toxin); Pseudomonas spp, including P. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis; Enterococcus spp., including E. jaecalis, E. jaecium; Clostridium spp., including C tetani (for example tetanus toxin and derivatives thereof), C botulinum (for example botulinum toxin and derivatives thereof, C difficile (for example Clostridium toxins A or B and derivatives thereof); Bacillus spp., including B. anthracis (for example botulinum toxin and derivatives thereof); Corynebacterium spp., including C diphtheriae (for example diphtheria toxin and derivatives thereof); Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA, DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (for example OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC, DbpA, DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia spp., including C trachomatis (for example MOMP, heparin-binding proteins), C. pneumoniae (for example MONT, heparin-binding proteins), C psittaci; Leptospira spp., including L. interrogans; Treponema spp., including T. pallidum (for example the rare outer membrane proteins), T denticola, T hyodysenteriae; or species derived from parasites such as Plasmodium spp., including P. falciparum; Toxoplasma spp., including T. gondii (for example SAG2, SAG3, Yg34); Entamoeba spp., including E. histolytica; Babesia spp., including B. microti; Trypanosoma spp., including T cruzi; Giardia spp., including G. lamblia; Leshmania spp., including L. major; Pneumocystis spp., including P. carinii; Trichomonas spp., including T. vaginalis; Schisostoma spp., including S. mansoni, or species derived from yeast such as Candida spp., including C albicans; Cryptococcus spp., including C neoformans.

Examples of preferred specific antigens for M. tuberculosis are for example Th Ra, Th H9, Th Ra35, Th38-1 , Erd 14, DPV, MTI, MSL, mTTC2 and hTCC1 (WO 99/51748). Proteins for M tuberculosis also include fusion proteins and variants thereof where at least two, preferably three polypeptides of M. tuberculosis are fused into a larger protein.

Examples of preferred antigens for Chlamydia include for example the High Molecular Weight Protein (HWMP) (WO 99/17741 ), ORF3 (EP 366 412), and putative membrane proteins (Pmps). Other Chlamydia antigens of the vaccine formulation can be selected from the group described in WO 99/28475.

Other preferred bacterial vaccines comprise antigens derived from Streptococcus spp, including S. pneumoniae (for example capsular polysaccharides and conjugates thereof, Psa.A, PspA, streptolysin, choline-binding proteins) and the protein antigen Pneumolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins et al., Microbial Pathogenesis, 25, 337- 342), and mutant detoxified derivatives thereof (WO 90/06951).

Cancer vaccines

A "cancer antigen" or "tumor antigen" as used herein is a compound, such as a peptide, associated with a tumor or cancer cell surface and which is capable of provoking an immune response when expressed on the surface of an antigen presenting cell in the context of an MHC molecule. Cancer antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells, for example, as described in Cohen, et al., 1994, Cancer Research, 54:1055, by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens. Cancer antigens include antigens that are recombinately an immunogenic portion of or a whole tumor or cancer. Such antigens can be isolated or prepared recombinatly or by any other means known in the art.

The formulations may also contain a tumour antigen and be useful for the immunotherapeutic treatment of cancers. For example, the adjuvant formulation finds utility with tumour rejection antigens such as those for prostate, breast, colorectal, lung, pancreatic, renal or melanoma cancers. Exemplary antigens include MAGE 1 and MAGE 3 or other MAGE antigens (for the treatment of melanoma), PRAME, BAGE, or GAGE (Robbins and Kawakami, 1996, Current Opinions in Immunology .8, pps 628-636; Van den Eynde et al., International Journal of Clinical & Laboratory Research (submitted 1997); Correale et al. (1997), Journal of the National Cancer Institute 89, p293. Indeed these antigens are expressed in a wide range of tumour types such as melanoma, lung carcinoma, sarcoma and bladder carcinoma. Other tumour-specific antigens are suitable for use with the adjuvants of the present invention and include, but are not restricted to tumour-specific gangliosides, Prostate specific antigen (PSA) or Her-2/neu, KSA (GA733), PAP, mammaglobin, MUC-1 , carcinoembryonic antigen (CEA). Accordingly in one aspect of the present invention there is provided a vaccine comprising an adjuvant composition according to the invention and a tumour rejection antigen.

It is a particularly preferred aspect of the present invention that the vaccines comprise a tumour antigen; such vaccines are surprisingly potent in the therapy of cancer such as prostrate, breast, colorectal, lung, pancreatic, renal, ovarian or melanoma cancers.

Accordingly, the formulations may contain tumour-associated antigen, as well as antigens associated with turnour-support mechanisms (e.g. angiogenesis, tumour invasion). Additionally, antigens particularly relevant for vaccines in the therapy of cancer also comprise Prostate-specific membrane antigen (PSMA), Prostate Stem Cell Antigen (PSCA), tyrosinase, survivin, NY-ES01 , prostase, PS108 (WO 98/50567), RAGE, LAGE, HAGE. Additionally said antigen may be a self peptide hormone such as whole length Gonadotrophin hormone releasing honnone (GnRH, WO 95/20600), a short 10 amino acid long peptide, useful in the treatment of many cancers, or in immunocastration.

It is foreseen that compositions of the present invention will be used to formulate vaccines containing antigens derived from Borrelia sp. Vaccines of the present invention may be used for the prophylaxis or therapy of allergy. Such vaccines would comprise allergen specific (for example Der p 1) and allergen non-specific antigens (for example peptides derived from human IgE, including but not restricted to the stanworth decapeptide (EP 0 477 231 B1)).

Vaccines of the present invention may also be used for the prophylaxis or therapy of chronic disorders others than allergy, cancer or infectious diseases. Such chronic disorders are diseases such as atherosclerosis, and Alzheimer. Antigens relevant for the prophylaxis and the therapy of patients susceptible to or suffering from Alzheimer neurodegenerative disease are, in particular, the N terminal 39 -43 amino acid fragment (AP) of the amyloid precursor protein and smaller fragments (WO 99/27944).

Each of the foregoing lists is illustrative, and is not intended to be limiting. The disclosures of each of foregoing references disclosing antigens and diseases or conditions are incorporated herein by reference.

Gamma-delta T cells activating compounds In another embodiment of the invention, methods of inducing a humoral immune response or inducing the production of antibodies are provided. In this aspect of the invention, a composition comprising an antigen of interest and at least one phosphoantigen compound is administered to an individual in an amount effective to induce the production of antibodies by said individual or induce a humoral immune response in said individual. The antigen of interest can be defined as any substance against which specific antibodies are desired. Non-limiting examples of such substances are peptides, polypeptides, proteins, tumor cells, tumor antigens, viruses, bacterial cells, parasites, haptens, or chemical entities (e.g., pharmaceutical drugs or drug metabolites thereof). The term "specific antibody" or "specific antibodies" is used in this application to indicate an antibody or antibodies that bind to an antigen of interest via the antigen combining site of said antibodies. In the context of this aspect of the invention, the term "individual" includes but is not limited to animals of mammalian origin. Mammalian species which benefit from the disclosed methods include but and are not limited to, humans, apes, chimpanzees, orangutans and monkeys, preferably New World Monkeys and Old World Monkeys.

The terms "γδ T lymphocyte activating compound", "γδ T cell activating compound", "γδ T lymphocyte activator" and "γδ T cell activator", used interchangeably, designate a molecule which can activate γδ T lymphocytes. Preferably the "γδ T lymphocyte activating compound" is a "Vγ9Vδ2 T lymphocyte activating compound", used herein interchangeably with the terms "Vγ9Vδ2 T cell activating compound", "Vγ9Vδ2 T lymphocyte activator" and "Vγ9Vδ2 T cell activator", designating a molecule which can activate Vγ9Vδ2 T cells. The activator may by of various nature, such as a peptide, lipid, small molecule, etc. It may be a purified or otherwise artificially produced (e.g., by chemical synthesis, or by microbiological process) endogenous ligand, or a fragment or derivative thereof, or an antibody having substantially the same antigenic specificity. The activator is most preferably a synthetic chemical compound capable of selectively activating Vγ9Vδ2 T lymphocytes. Selective activation of Vγ9Vδ2 T lymphocytes indicates that the compound has a selective action towards specific cell populations, and essentially does not activate other T cell sub-types, such as Vδ1 T cells. Such selectivity, as disclosed in the present application, suggests that preferred compounds can cause a selective or targeted activation of, proliferation of or other biological activity of Vγ9Vδ2 T lymphocytes.

Preferably a γδ T lymphocyte activator is a compound capable of regulating the activity of a γδ T cell, preferably a Vγ9Vδ2 T cell, in an individual, for example in a human or a non- human primate. In one example, γδ T lymphocyte activation can be assessed by administering a compound to an individual (human or non-human primate) and assessing activation or proliferation of Vγ9Vδ2 T lymphocytes. In an exemplary protocol expansion of the Vγ9Vδ2 T cell population is assessed: a candidate γδ T lymphocyte activator is administered to a non-human primate such as a cynomolgus monkey by intravenous infusion (one administration by slow infusion, 50 ml over 30 minutes) in combination with IL-2 (0.9 million units twice daily by subcutaneous injection for 5 days); peripheral γδ lymphocytes are analysed by flow cytometry on total monkey blood, after double staining with anti- CD3-PE antibody and anti-Vgamma9-FITC antibodies and/or anti Vd2 antibodies, and cells are counted by flow cytometry. Peak expansion of the Vγ9Vδ2 T cell population is observed between days 3 and 8, generally at about days 4-6 after administration of the γδ T lymphocyte activator.

Any other suitable tests can be used to assess cell proliferation. Assessment of proliferation or peripheral γδ lymphocytes can generally be analyzed by flow cytometry on total blood (for example total blood obtained from a monkey), after double staining with anti-CD3-PE antibody and anti-Vgamma9-FITC antibodies and/or anti Vd2 antibodies (CD3-PE : SP34 clone, BD Biosciences Pharmingen, Le Pont de Claix, France). Anti Vgamma 9, clone 7B6 is a monoclonal raised to human Vgamma 9 but that cross-reacts with cynomolgus monkey cells. It is purified by affinity chromatography on protein A and coupled to FITC. 50μl monkey blood is incubated 15 min at RT with 5μl anti-CD3-PE and 6μl anti-delta2-FITC or 10μl anti-gamma9-FITC antibodies. Antibodies are washed with 3ml 1X PBS, centrifuged for 4 min at 1300rpm at RT and supernatant is discarded. Red cells are lysed with the OptiLyse C reagent (Immunotech-Beckman-Coulter, Marseilles, France) according to the manufacturer's instructions. At the final step, stained white blood cells are recovered by centrifugation and resuspended in 300μl PBS + 0.2% PFA. Immediately before analysis, 50μl calibrated Flow CountTM Fluorospheres (Immunotech- Beckman-Coulter, Marseilles, France) are added to the cells for absolute number counting of the populations of interest.

Preferably a γδ T lymphocyte activator is a compound capable of regulating the activity of a γδ T cell in a population of γδT cell clones in culture. The γδ T lymphocyte activator is more preferably capable of regulating the activity of a γδ T cell population of γδT cell clones in a at millimolar concentration, preferably when the γδ T cell activator is present in culture at a concentration of less than 100 mM. In one example, cytokine production or release is assessed. Vg9Vd2 cells are known producers of TNFα and IFNγ in vitro upon phosphoantigen administration. Shortly after BrHPP treatment samples of sera are collected from an individual and are assayed by ELISA specific for TNFα or IFNγ.

Regulating the activity of a γδ T cell can be assessed by any suitable means, preferably by assessing cytokine secretion, most preferably TNF-α secretion as described herein.

Methods for obtaining a population of pure γδ T cell clones is described in Davodeau et al, ((1993) J. Immunology 151(3): 1214-1223) and Moreau et al, ((1986) J. Clin. Invest. 78:874), the disclosures of which are incorporated herein by reference. Preferably the γδ T cells are of the Vγ9Vδ2 type.

Cytokine secretion can be assessed using any appropriate in vitro assay, or those provided in the examples herein. For example, cytokine secretion can be determined according to the methods described in Espinosa et al. (J. Biol. Chem., 2001 , Vol. 276, Issue 21 , 18337-18344), describing measurement of TNF-α release in a bioassay using TNF-α-sensitive cells. Briefly, 10⁴ YδT cells/well are incubated with stimulus plus 25 units of IL2/well in 100 μl of culture medium during 24 h at 37 ⁰C. Then, 50 μl of supernatant are added to 50 μl of WEHI cells plated at 3 x 10⁴ cells/well in culture medium plus actinomycin D (2 μg/ml) and LiCI (40 mM) and incubated for 20 h at 37 ⁰C. Viability of the TNF-α-sensitive cells are measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazoiium bromide assay. 50 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (Sigma; 2.5 mg/ml in phosphate-buffered saline) per well are added, and after 4 h of incubation at 37 ⁰C, 50 μl of solubilization buffer (20% SDS, 66% dimethyl formamide, pH 4.7) are added, and absorbance (570 nm) is measured. Levels of TNF-α release are then calculated from a standard curve obtained using purified human rTNF-α (PeproTech, Inc., Rocky Hill, NJ). TNF-α detection can also be carried out according to the manufacturer's instructions (kit ref 1121 , Immunotech-Beckman Coulter). Interferon-Y released by activated T cells is measured by a sandwich enzyme-linked immunosorbent assay. 5 x 10⁴ YST cells/well are incubated with stimulus plus 25 units of IL2/well in 100 μl of culture medium during 24 h at 37 ⁰C. Then, 50 μl of supernatant is harvested for enzyme-linked immunosorbent assay using mouse monoclonal antibodies (Biosource International, California).

For methods of inducing a humoral immune response in an individual, the compositions to be administered contain one or more phosphoantigen compounds at a concentration that provides about: 0.10 mg to 1000 mg; 0.40 mg to 500 mg; or 0.40 mg to 40 mg of compounds per kilogram of body weight of said individual. Other embodiments in methods for inducing a humoral immune response in an individual provide compositions that comprise phosphoantigen compounds at a concentration that provides at least: 0.10 mg; 0.25 mg; 0.40 mg, 4.0 mg; or 40 mg of compound per kilogram of body weight of said individual. Compositions used for the induction of humoral immune responses can, optionally, further comprise other adjuvants such as aluminum salts (e.g. aluminum hydroxides or aluminum phosphates). Additional adjuvants include, and are not limited to, calcium phosphate, endotoxin monophosphoryl lipid A, exotoxins cholera toxin, E. coli heat-labile toxin, pertussis toxin, muramyl dipeptide (MDP), Freund's Incomplete Adjuvant (IFA), MF59, SAF, liposomes, biodegradable microspheres, saponins (QS-21), nonionic block copolymers, muramyl peptide analogues, polyphosphazene and synthetic polynucleotides. Additional exemplary adjuvants for use in formulating compositions for use in the subject invention are listed in A Compendium of Vaccine Adjuvants and Excipients (2nd Edition), Frederick R. Vogel, Michael F. Powell, and Carl R. Alving (available through the National Institute of Allergy and Infectious Diseases, Bethesda MD or on the worldwide web at niaid. nih.gov/daids/vaccine/pdf/compendium.pdf) which is hereby incorporated by reference in its entirety, particularly with respect to the listed adjuvants.

Phosphoantigen compounds useful as adjuvants for the stimulation of a humoral immune response comprise the compounds of formula (I) :

Formula (I) wherein Cat+ represents one (or several, identical or different) organic or mineral cation(s)

(including protons); m is an integer from 1 to 3;

B is O, NH, or any group capable of being hydrolyzed; Y = O^"Cat+, a C₁-C₃ alkyl group, a group -A-R₀, or a radical selected from the group consisting of a nucleoside, an oligonucleotide, a nucleic acid, an amino acid, a peptide, a protein, a monosaccharide, an oligosaccharide, a polysaccharide, a fatty acid, a simple lipid, a complex lipid, a folic acid, a tetrahydrofolic acid, a phosphoric acid, an inositol, a vitamin, a co-enzyme, a flavonoid, an aldehyde, an epoxide and a halohydrin; A is O, NH, CHF, CF₂ or CH₂; and,

R₀ and R can be the same or different and are a linear, branched, or cyclic, aromatic, non- aromatic, saturated C₁-C₅₀ Or unsaturated C₁-C₅₀ hydrocarbon group, optionally interrupted by at least one heteroatom, wherein said hydrocarbon group comprises an alkyl, an alkylenyl, or an alkynyl, preferably an alkyl or an alkylene, which can be substituted by one or several substituents selected from the group consisting of : an alkyl, an alkylenyl, an alkynyl, an epoxyalkyl, an aryl, an heterocycle, an alkoxy, an acyl, an alcohol, a carboxylic group (-COOH), an ester, an amine, an amino group (-NH₂), an amide (- CONH₂), an imine, a nitrile, an hydroxyl (-OH), a aldehyde group (-CHO), an halogen, an halogenoalkyl, a thiol (-SH), a thioalkyl, a sulfone, a sulfoxide, and a combination thereof.

In a particular embodiment, the substituents as defined above are substituted by at least one of the substituents as specified above.

Preferably, the substituents are selected from the group consisting of : an (Ci-C₆)alkyl, an (C₂-C₆)alkylenyl, an (C₂-C₆)alkynyl, an (C₂-C₆)epoxyalkyl, an aryl, an heterocycle, an (C₁- C₆)alkoxy, an (C₂-C₆)acyl, an (C_rC₆)alcohol, a carboxylic group (-COOH), an (C₂-C₆)ester, an (CrCeJamine, an amino group (-NH₂), an amide (-CONH₂), an (CrC₆)imine, a nitrile, an hydroxyl (-OH), a aldehyde group (-CHO), an halogen, an (CrC₆)halogenoalkyl, a thiol (-SH), a (CrCβJthioalkyl, a (CrCeJsulfone, a (d-CeJsulfoxide, and a combination thereof.

More preferably, the substituents are selected from the group consisting of : an (C₁- C₆)alkyl, an (C₂-C₆)epoxyalkyl, an (C₂-C₆)alkylenyl, an (C₁-C₆JaIkOXy, an (C₂-C₆)acyl, an (Ci-C₆)alcohol, an (C₂-C₆)ester, an (CrC₆)amine, an (C_rC₆)imine, an hydroxyl, a aldehyde group, an halogen, an (C_rC₆)halogenoalkyl, and a combination thereof.

Still more preferably, the substituents are selected from the group consisting of : an (C₃- C₆)epoxyalkyl, an (CrC₃)alkoxy, an (C₂-C₃)acyl, an (C₁-C₃JaIcOhOl, an (C₂-C₃)ester, an (CrC^amine, an (C₁-C₃)JmJnC, an hydroxyl, an halogen, an (CrC₃)halogenoalkyl, and a combination thereof, and a combination thereof. Preferably, R is a (C₃-C₂₅)hydrocarbon group, more preferably a (C₅-C₁₀)hydrocarbon group.

In the context of the present invention, the term "alkyl" more specifically means a group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, te/f-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl and the other isomeric forms thereof. (CrQOalkyl more specifically means methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl and the other isomeric forms thereof. (C_rC₃)alkyl more specifically means methyl, ethyl, propyl, or isopropyl. The term "alkenyl" refers to an alkyl group defined hereinabove having at least one unsaturated ethylene bond and the term "alkynyl" refers to an alkyl group defined hereinabove having at least one unsaturated acetylene bond. (C₂-C₆)alkylene includes a ethenyl, a propenyl (1-propenyl or 2-propenyl), a 1- or 2- methylpropenyl, a butenyl (1- butenyl, 2-butenyl, or 3-butenyl), a methylbutenyl, a 2-ethylpropenyl, a pentenyl (1- pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl), an hexenyl (1-hexenyl, 2-hexenyl, 3- hexenyl, 4-hexenyl, 5-hexenyl), and the other isomeric forms thereof. (C₂-C₆)alkynyl includes ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2- pentynyl, 3-pentynyI, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl and the other isomeric forms thereof.

The term "epoxyalkyl" refers to an alkyl group defined hereinabove having an epoxide group. More particularly, (C₂-C₆)epoxyalkyl includes epoxyethyl, epoxypropyl, epoxybutyl, epoxypentyl, epoxyhexyl and the other isomeric forms thereof. (C₂-C₃)epoxyalkyl includes epoxyethyl and epoxypropyl.

The "aryl" groups are mono-, bi- or tri-cyclic aromatic hydrocarbons having from 6 to 18 carbon atoms. Examples include a phenyl, α-naphthyl, β-naphthyl or anthracenyl group, in particular.

"Heterocycle" groups are groups containing 5 to 18 rings comprising one or more heteroatoms, preferably 1 to 5 endocyclic heteroatoms. They may be mono-, bi- or tri- cyclic. They may be aromatic or not. Preferably, and more specifically for R₅, they are aromatic heterocycles. Examples of aromatic heterocycles include pyridine, pyridazine, pyrimidine, pyrazine, furan, thiophene, pyrrole, oxazole, thiazole, isothiazole, imidazole, pyrazole, oxadiazole, triazole, thiadiazole and triazine groups. Examples of bicycles include in particular quinoline, isoquinoline and quinazoline groups (for two 6-membered rings) and indole, benzimidazole, benzoxazole, benzothiazole and indazole (for a 6- membered ring and a 5-membered ring). Nonaromatic heterocycles comprise in particular piperazine, piperidine, etc.

"Alkoxy" groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an -O- (ether) bond. (C_rC₆)alkoxy includes methoxy, ethoxy, propyloxy, butyloxy, pentyloxy, hexyloxy and the other isomeric forms thereof. (C_rC₃)alkoxy includes methoxy, ethoxy, propyloxy, and isopropyloxy. "Alcyl" groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an -CO- (carbonyl) group. (C₂-C₆)acyl includes acetyl, propylacyl, butylacyl, pentylacyl, hexylacyl and the other isomeric forms thereof. (C₂-C₃)acyl includes acetyl, propylacyl and isopropylacyl.

"Alcohol" groups correspond to the alkyl groups defined hereinabove containing at least one hydroxyl group. Alcohol can be primary, secondary or tertiary. (C₁-C₆JaIcOhOl includes methanol, ethanol, propanol, butanol, pentanol, hexanol and the other isomeric forms thereof. (C₁-C₃JaIcOhOl includes methanol, ethanol, propanol and isopropanol.

"Ester" groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an -COO- (ester) bond. (C₂-C₆)ester includes methylester, ethylester, propylester, butylester, pentylester and the other isomeric forms thereof. (C₂-C₃)ester includes methylester and ethylester.

"Amine" groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an -N- (amine) bond. (Ci-C₆)amine includes methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine and the other isomeric forms thereof. (CrC₃)amine includes methylamine, ethylamine, and propylamine.

"Imine" groups correspond to the alkyl groups defined hereinabove having a (-C=N-) bond. (CrCβJimine includes methylimine, ethylimine, propylimine, butylimine, pentylimine, hexylimine and the other isomeric forms thereof. (CrC₃)imine includes methylimine, ethylimine, and propylimine.

The halogen can be Cl, Br, I, or F, more preferably Br or F.

"Halogenoalkyl" groups correspond to the alkyl groups defined hereinabove having at least one halogen. The groups can be monohalogenated or polyhalogenated containing the same or different halogen atoms. For example, the group can be an trifluoroalkyl (CF₃- R). (CrCeJhalogenoalkyl includes halogenomethyl, halogenoethyl, halogenopropyl, halogenobutyl, halogenopentyl, halogenohexyl and the other isomeric forms thereof. (C₁- C₃)halogenoalkyl includes halogenomethyl, halogenoethyl, and halogenopropyl.

"Thioalkyl" groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an -S- (thioether) bond. (Ci-C₆)thioalkyl includes thiomethyl, thioethyl, thiopropyl, thiobutyl, thiopentyl, thiohexyl and the other isomeric forms thereof. (C₁-

C₃)thioalkyl includes thiomethyl, thioethyl, and thiopropyl.

"Sulfone" groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an -SOO- (sulfone) bond. (C_rC₆)sulfone includes methylsulfone, ethylsulfone, propylsulfone, butylsulfone, pentylsulfone, hexylsulfone and the other isomeric forms thereof. (d-CjJsulfone includes methylsulfone, ethylsulfone and propylsulfone.

"Sulfoxyde" groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an -SO- (sulfoxide) group. (Ci-C₆)sulfoxide includes methylsulfoxide, ethylsulfoxide, propylsulfoxide, butylsulfoxide, pentylsulfoxide, hexylsulfoxide and the other isomeric forms thereof. (CrC₃)sulfoxide includes methylsulfoxide, ethylsulfoxide, propylsulfoxide and isopropylsulfoxide.

"Heteroatom" denotes N, S, or O.

"Nucleoside" includes adenosine, thymine, uridine, cytidine and guanosine.

In a particular embodiment, the hydrocarbon group is a cycloalkylenyl such as a cyclopentadiene or a phenyl, or an heterocycle such as a furan, a pyrrole, a thiophene, a thiazole, an imidazole, a triazole, a pyridine, a pyrimidine, a pyrane, or a pyrazine. Preferably, the cycloalkylenyl or the heterocycle is selected from the group consisting of a cyclopentadiene, a pyrrole or an imidazole. In a preferred embodiment, the cycloalkylenyl or the heterocycle is sustituted by an alcohol. Preferably, said alcohol is a (C₁-C₃JaIcOhOl.

In an other embodiment, the hydrocarbon group is an alkylenyl with one or several double bonds. Preferably, the alkylenyl group has one double bond. Preferably, the alkylenyl group is a (C₃-Ci ₀)alkylenyl group, more preferably a (C₄-C₇)alkylenyl group. Preferably, said alkylenyl group is substituted by at least one functional group. More preferably, the functional group is selected from the group consisting of an hydroxy, an (CrC₃)alkoxy, an aldehyde, an (C₂-C₃)acyl, or an (C₂-C₃)ester. In a more preferred embodiment, the hydrocarbon group is butenyl substituted by a group -CH₂OH. Optionally, said alkenyl group can be the isoform trans (E) or cis (Z), more preferably a trans isoform (E). In a most preferred embodiment, the alkylenyl group is the (E)-4-hydroxy-3-methyl-2-butenyl. In an other preferred embodiment, the alkylenyl group group is an isopentenyl, an dimethylallyl or an hydroxydimethylallyl. In an additional embodiment, the hydrocarbon group is an alkyl group substituted by an acyl. More preferably, the hydrocarbon group is an (C₄-C₇)alkyl group substituted by an (C_rC₃)acyl.

In a further preferred embodiment, R is selected from the group consisting of :

1)

wherein n is an integer from 2 to 20, R₁ is a (C_rC₃)alkyl group, and R₂ is an halogenated (C_rC₃)alkyl, a (C₁-C₃)alkoxy-(C_rC₃)alkyl, an halogenated (C₂-C₃)acyl or a (C_rC₃)alkoxy- (C₂-C₃)acyl. Preferably, R₁ is a methyl or ethyl group, and R₂ is an halogenated methyl (- CH₂-X, X being an halogen), an halogenated (C₂-C₃)acetyl, or (C₁-C₃)BIkOXy- acetyl. The halogenated methyl or acetyl can be mono-, di-, or tri-halogenated. Preferably, n is an integer from 2 to 10, or from 2 to 5. In a more preferred embodiment, n is 2. In a most preferred embodiment, n is 2, R₁ is a methyl and R₂ is an halogenated methyl, more preferably a monohalogenated methyl, still more preferably a bromide methyl. In a particularly preferred embodiment, n is 2, R₁ is a methyl, R2 is a methyl bromide. In a most preferred embodiment, R is 3-(bromomethyl)-3-butanol-1-yl,

2)

wherein n is an integer from 2 to 20, and R₁ is a methyl or ethyl group. Preferably, n is an integer from 2 to 10, or from 2 to 5. In a more preferred embodiment, n is 2 and R1 is a methyl, and

3)

R, ,R<

— C — W = C

R, R₆ wherein R₃, R₄, and R₅ , identical or different, are a hydrogen or (d-C₃)alkyl group, W is -

CH- or -N-, and R₆ is an (C₂-C₃)acyl, an aldehyde, an (CrC^alcohol, or an (C₂-C₃)ester. More preferably, R₃ and R₅ are a methyl and R₄ is a hydrogen. More preferably, R₆ is - CH₂-OH, -CHO, -CO-CH₃ or -CO-OCH₃. Optionally, the double-bond between W and C is in conformation trans (E) or cis (Z). More preferably, the double-bond between W and C is in conformation trans (E).

The group Y can allow to design a prodrug. Therefore, Y is enzymolabile group which can be cleaved in particular regions of the subject. The group Y can also be targeting group. In a preferred embodiment, Y is O^"Cat+, a group -A-R₀, or a radical selected from the group consisting of a nucleoside, a monosaccharide, an epoxyde and a halohydrin. Preferably, Y is an enzymolabile group. Preferably, Y is O^'Cat÷, a group -A-R₀, or a nucleoside. In a first preferred embodiment, Y is O^"Cat+. In a second preferred embodiment, Y is a nucleoside.

In a preferred embodiment, Cat^* is H⁺, Na⁺, NH₄ ⁺, K⁺, Li⁺, (CH₃CH₂)₃NH⁺.

In a preferred embodiment, A is O, CHF, CF₂ or CH₂. More preferably, A is O or CH₂.

In a preferred embodiment, B is O or NH. More preferably, B is O.

In a preferred embodiment, m is 1 or 2. More preferably, m is 1.

In one particular embodiment, phosphoantigen compounds comprise the compounds of formula (II):

in which X is an halogen (preferably selected from I₁ Br and Cl), B is O or NH, m is an integer from 1 to 3, R1 is a methyl or ethyl group, Cat+ represents one (or several, identical or different) organic or mineral cation(s) (including the proton), and n is an integer from 2 to 20, A is O, NH, CHF, CF₂ or CH₂, and Y is O^"Cat+, a nucleoside, or a radical -A- R₀, wherein R₀ is selected from the group of 1), 2) or 3). Preferably, Y is O^"Cat+, or a nucleoside. More preferably, Y is O^"Cat+. Preferably, R1 is a methyl. Preferably, A is O or CH₂. More preferably, A is O. Preferably, n is 2. Preferably, X is a bromide. Preferably, B is O. Preferably, m is 1 or 2. More preferably, m is 1. For example, phosphoantigen compounds comprise the compounds of formula (III) or (IV)

wherein X, R1, n, m and Y have the aforementioned meaning.

In one preferred embodiment, phosphoantigen compounds comprise the compounds of formula (V):

in which X is an halogen (preferably selected from I, Br and Cl), R1 is a methyl or ethyl group, Cat+ represents one (or several, identical or different) organic or mineral cation(s) (including the proton), and n is an integer from 2 to 20. Preferably, R1 is a methyl. Preferably, n is 2. Preferably, X is a bromide.

In a most preferred embodiment, phosphoantigen compounds comprise the compound of formula (Vl):

Preferably x Cat+ is 1 or 2 Na⁺

In another most preferred embodiment, phosphoantigen compounds comprise the compound of formula (VII):

Preferably x Cat+ is 1 or 2 Na⁺.

Specific examples of compounds include, and are not limited to:

(E)1-pyrophosphonobuta-1,3-diene;

(E)1-pyrophosphonopenta-1 ,3-diene;

(E)1-pyrophosphono-4-methylpenta-1 ,3-diene;

(E,E)1-pyrophosphono-4,8-dimethylnona-1 ,3,7-triene; (E,E,E)1-pyrophosphono-4,8,12-trimethyltrideca-1 ,3,7,11-tetraene;

(E,E)1-triphosphono-4,8-dimethylnona-1,3,7-triene;

4- triphosphono-2-methylbutene; α,β-di-[3-methylpent-3-enyl]-pyrophosphonate;

1-pyrophosphono-3-methylbut-2-ene; α,γ-di-[3-methylbut-2-enyl]-triphosphonate; α,β-di-[3-methylbut-2-enyl]-pyrophosphonate; allyl-pyrophosphonate; allyl-triphosphonate; α,γ-di-allyl-pyrophosphonate; α,β-di-allyl-triphosphonate;

(E,E)4-[(5'-pyrophosphono-6'-methyl-penta-2',4'-dienyloxymethyl)-phenyl]- phenylmethanone;

(E,E)4-[(5'-triphosphono-6'-methyl-penta-2',4'-dienyloxymethyl)-phenyl]-phenyl- methanone; (E,E,E)[4-(9'-pyrophosphono-2',6'-dimethyl-nona-2',6',8'-trienyloxymethyl)-phenyl]-phenyl- methanone;

(E,E,E)[4-(9'-pyrophosphono-2',6',8'-trimethyl-nona-2',6',8'-trienyloxymethyl)-phenyl]- phenyl-methanone;

5-pyrophosphono-2-methypentene; 5-triphosphono-2-methypentene; α,γ-di-[4-methylpent-4-enyl]-triphosphonate;

5-pyrophosphono-2-methypent-2-ene;

5-triphosphono-2-methypent-2-ene;

9-pyrophosphono-2,6-dimethynona-2,6-diene; 9-triphosphono-2,6-dimethynona-2,6-diene; α,γ-di-[4,8-dimethylnona-2,6-dienyl]-triphosphonate;

4-pyrophosphono-2-methybutene;

4-methyl-2-oxa-pent-4-enyloxymethylphosphoantigen; 4-methyl-2-oxa-pent-4-enyloxymethyltriphosphate; α,β-di-[4-methyl-2-oxa-pent-4-enyloxymethyl]-phosphoantigen; α,γ-di-[4-methyl-2-oxa-pent-4-enyloxymethyl]-triphosphate;

3-(halomethyl)-3-butanol-1-yl-diphosphate;

3-(halomethyl)-3-pentanol-1-yl-diphsophate; 4-(halomethyl)-4-pentanol-1 -yl-diphosphate;

4-(halomethyl)-4-hexanol-1-yl-diphosphate; δ-ChalomethyO-δ-hexanol-i-yl-diphosphate;

5-(halomethyl)-5-heptanol-1-yl-diphosphate;

6-(halomethyl)-6-heptanol-1 -yl-diphosphate; 6-(halomethyl)-6-octanol-1 -yl-diphosphate;

7-(halomethyl)-7-octanol-1-yl-diphosphate;

7-(halomethyl)-7-nonanol-1-yl-diphosphate;

8-(halomethyl)-8-nonanol-1-yl-diphosphate;

8-(halomethyl)-8-decanol-1 -yl-diphosphate; 9-(halomethyl)-9-decanol-1 -yl-diphosphate;

9-(halomethyl)-9-undecanol-1-yl-diphosphate;

10-(halomethyl)-10-undecanol-1 -yl-diphosphate;

10-(halomethyl)-10-dodecanol-1 -yl-diphosphate;

11 -(halomethyl)-i 1 -dodecanol-1 -yl-diphosphate; 11 -(halomethyl)-i 1 -tridecanol-1 -yl-diphosphate;

12-(halomethyl)-12-tridecanol-1 -yl-diphosphate;

12-(halomethyl)-12-tetradecanol-1 -yl-diphosphate;

13-(halomethyl)-13-tetradecanol-1 -yl-diphosphate;

13-(halomethyl)-13-pentadecanol-1 -yl-diphosphate; 14-(halomethyl)-14-pentadecanol-1 -yl-diphosphate;

14-(halomethyl)-14-hexadecanol-1 -yl-diphosphate;

15-(halomethyl)-15-hexadecanol-1 -yl-diphosphate;

15-(halomethyl)-15-heptadecanol-1 -yl-diphosphate;

16-(halomethyl)-16-heptadecanol-1 -yl-diphosphate; 16-(halomethyl)-16-octadecanol-1 -yl-diphosphate;

17-(halomethyl)-17-octadecanol-1 -yl-diphosphate;

17-(halomethyl)-17-nonadecanol-1 -yl-diphosphate; 18-(halomethyl)-18-nonadecanol-1 -yl-diphosphate;

18-(halomethyl)-18-eicosanol-1 -yl-diphosphate;

19-(halomethyl)-19-eicosanol-1 -yl-diphosphate;

19-(halomethyl)-19-heneicosanol-1 -yl-diphosphate; 20-(haiomethyl)-20-heneicosanol-1 -yl-diphosphate;

20-(halomethyl)-20-docosanol-1-yl-diphosphate;

21 -(halomethyl)-21-docosanol-1 -yl-diphosphate;

21 -(halomethyl)-21-tricosanol-1 -yl-diphosphate;

3-(bromomethyl)-3-butanol-1 -yl-diphosphate (BrHPP); 5-bromo-4-hydroxy-4-methylpentyl pyrophosphonate (CBrHPP);

3-(iodomethyl)-3-butanol-1 -yl-diphosphate (IHPP);

3-(chloromethyl)-3-butanol-1 -yl-diphosphate (CIHPP);

3-(bromomethyl)-3-butanol-1-yl-triphosphate (BrHPPP);

3-(iodomethyl)-3-butanol-1-yl-triphosphate (IHPPP); α,γ-di-[3-(bromomethyl)-3-butanol-1-yl]-triphosphate (diBrHTP); or α,γ-di-[3-(iodomethyl)-3-butanol-1-yl]-triphosphate (dilHTP), wherein halo- is any halogen atom (preferably I, Br, or Cl).

Unless other wise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Particularly, the following patent applications are hereby incorporated by reference in their entireties (including all figures, formulae, amino acid sequences and polynucleotide sequences): United States Patent Application 10/494,537, filed April 30, 2004; International Application PCT/EP02/12101 , filed October 30, 2002; European Patent Application EP 01870234.0, filed October 31 , 2001 , U.S Patent No 6,660,723 and European Patent No. EP 1 109 817 Bl The materials, methods, and examples are illustrative only and not intend to be limiting. Other features and advantages of the invention will be apparent from the following drawings, detailed description, and from the claims. EXAMPLES

Example 1

Vγ9Vδ2 T cell activation by BrHPP compound and EC50 as determined using a cytokine release assay

The synthesis and characterization of trisodium (r,s)-3-(bromomethyl)-3-butanol-1-yl- diphosphate BrHPP has been described previously and can be carried out as described in Espinosa, E., Belmant, C, Pont, F., Luciani, B., Poupot, R., Romagne, F., Brailly, H., Bonneville, M., And Fournie, J. J. (2001a). Chemical Synthesis And Biological Activity Of Bromohydrin Pyrophosphate, A Potent Stimulator Of Human Gamma Delta T Cells. J Biol Chem 276, 18337-18344.

Activation of human Vγ9Vδ2 T cells is assessed by measuring TNFα production by Vγ9Vδ2 T cells. Cells (primary polyclonal human Vγ9Vδ2 T cells which have been expanded in vitro and stored frozen at day 12-15 of expansion) are thawed and rinsed twice and centrifuged. Upon elimination of supernatant and resuspension of cells, the cells are incubated for 24h at 37°C in the presence of IL2 100 IU/ml (final concentration). The cells are washed and centrifuged, following which the supernatant is eliminated and the cells are resuspended and adjusted to the adequate final concentration. The cells are added to the wells of a 96-well plate. To one row of wells is added a dilution series of 3- (bromomethyl)-3-butanol-1-yl-diphosphate (BrHPP). Full plates are incubated 24 hours at 37°C for stimulation of the Vγ9Vδ2 cells with the BrHPP compound. After the incubation, 100 μl of culture supernatant is taken for TNFα dosage. Measurement of the released TNFα dosage is performed as described by the manufacturer's instruction in the TNFα enzyme immunoassay kit (ref. 11121 , lmmunotech - Beckman Coulter). OD at 405nm is read, the OD being proportional to the concentration of released TNFα in the culture supernatant. The data are processed with the Excel software to compare concentration of test compound versus concentration of TNFα and for the calculation of the EC50.

The BrHPP compound demonstrated a potent activation of human Vγ9Vδ2 T cells compared to control. The assessment of bioactivity of the BrHPP compound using a TNFα release assay was repeated for several batches of the compound. The in vitro EC50 for BrHPP was found to range from about 15nM to 38nM. Example 2 Evaluation of BrHPP as an adjuvant for HIV antigens

In this example, the effect of a phosphoantigen (BrHPP) on immune responses induced by the recombinant HIV protein Tat was examined. Cynomolgus monkeys were immunized twice (IM route) at 4-week intervals. 3 doses of BrHPP were tested: 0.4 mg/kg, 4 mg/kg and 40 mg/kg.

To briefly set forth the protocol, thirty-two (32) Cynomolgus macaques (male animals weighing 3-4 kg each) were randomized into seven (7) groups of 3-5 animals and immunized according to the following protocol:

2 intramuscular (IM) injections with 2 distinct syringes were given at a 4 week interval. Injections contained Tat protein with BrHPP (groups 2 to 6), and for purposes of comparison Tat and PBS (group 7) and Tat and aluminium hydroxide AIOOH (Group 1). IM injections were performed in the same anatomical site. 1 ml of blood for serology was analysed two weeks after the second injection. Groups: Tat Protocol

- Group 1 : Tat 10μg + AIOOH 300μg 3 monkeys

- Group 2 : Tat 10μg + BrHPP 40mg/kg 5 monkeys

- Group 3 : Tat 10μg + BrHPP 40mg/kg 5 monkeys

- Group 4 : Tat 10μg + BrHPP 0.4mg/kg 5 monkeys

- Group 5 : Tat 10μg + BrHPP 0.4mg/kg 5 monkeys

- Group 6 : Tat 10μg + BrHPP 4mg/kg 5 monkeys

- Group 7 : Tat 10μg + PBS 4 monkeys

Humoral Response

The antigen specific antibody responses raised in immunized animals were tested by standard ELISAs and expressed as ELISA titers (Iog10). Tat specific antibody titers were measured at two points prior to Tat administration - week 0 and week 10 - the latter being one week before this first Tat administration, and at week 17 (after two administrations of 10μg/ml of Tat. Results are shown in Figure 1.

The administration of 10μg of Tat in the absence of BrHPP resulted in only low levels of specific antibody (<2 Iog10). The addition of BrHPP into the vaccines noticeably increases the antibody responses. The 3 doses of BrHPP were active with 0.4 mg/kg being more active than 4 or 40 mg/kg, with up to a 2 log increase in antibody titers.

Claims

1. Use of an antigen and at least one phosphoantigen compound for the preparation of a medicament for enhancing a humoral immune response of an individual.

2. The use according to claim 1 , wherein said antigen is a peptide, polypeptide, protein, virus, bacterium, parasite.

3. The use according to claim 1 or 2, wherein said phosphoantigen composition modulates the function of a Vγ9Vδ2 T cell.

4. The use according to claim 1 or 2, wherein said phosphoantigen composition is capable of activating a Vγ9Vδ2 T cell upon administration to an individual.

5. The use according to claim 1 or 2, wherein said phosphoantigen composition induces cytokine production by a Vγ9Vδ2 T cell when brought into contact with a Vγ9Vδ2 T cell in culture.

6. The use according to any one of claims 1-4, wherein said composition comprises at least one phosphoantigen compound at a concentration that provides about 0.10 mg to

1000 mg of compound per kilogram of body weight of said individual.

7. The use according to any one of claims 1-4, wherein said composition comprises at least one phosphoantigen compound at a concentration that provides about 0.40 mg to 40 mg of compound per kilogram of body weight of said individual.

8. The use according to claim 1 or 2, wherein said composition comprises at least one phosphoantigen compound at a concentration that provides at least 0.10 mg of compound per kilogram of body weight of said individual.

9. The use according to claim 1 or 2, wherein said composition comprises at least one phosphoantigen compound at a concentration that provides at least 0.25 mg of compound per kilogram of body weight of said individual.

10. The use according to any one of preceding claims, wherein said individual is a human.

11. The use according to any one of preceding claims, wherein said phosphoantigen compound is of formula (I) :

B is O, NH, or any group capable of being hydrolyzed;

R₀ and R can be the same or different and are a linear, branched, or cyclic, aromatic, non- aromatic, saturated C₁-C₅₀ Or unsaturated C₁-C₅₀ hydrocarbon group, optionally interrupted by at least one heteroatom, wherein said hydrocarbon group comprises an alkyl, an alkylenyl, or an alkynyl, which can be substituted by one or several substituents selected from the group consisting of: an alkyl, an alkylenyl, an alkynyl, an epoxyalkyl, an aryl, an heterocycle, an alkoxy, an acyl, an alcohol, a carboxylic group (-COOH), an ester, an amine, an amino group (-NH₂), an amide (-CONH₂), an imine, a nitrile, an hydroxyl (-OH), a aldehyde group (-CHO), an halogen, an halogenoalkyl, a thiol (-SH), a thioalkyl, a sulfone, a sulfoxide, and combinations thereof.

12. The use according to claim 11 , wherein said phosphoantigen compound comprises the compounds of formula (II):

in which X is a halogen, B is O or NH, m is an integer from 1 to 3, R1 is a methyl or ethyl group, Cat+ represents at least one identical or different organic or mineral cation(s) or protons, n is an integer from 2 to 20, A is O, NH, CHF, CF₂ or CH₂, and Y is O^'Cat+, a nucleoside, or a radical -A-R₀, wherein R₀ is selected from the group of:

1)

OH

— (CH₂)_n — C — R₂

Ri wherein n is an integer from 2 to 20, R₁ is a (CrC₃)alkyl group, and R₂ is an halogenated

(CrCsJalkyl, a (C_rC3)alkoxy-(Ci-C₃)alkyl, an halogenated (C₂-C₃)acyl or a (C_rC₃)alkoxy- (C₂-C₃)acyl;

2)

wherein n is an integer from 2 to 20, and R₁ is a methyl or ethyl group; and

3)

wherein R₃, R₄, and R₅ , identical or different, are a hydrogen or (C_rC₃)alkyl group, W is - CH- or -N-, and R₆ is an (C₂-C₃)acyl, an aldehyde, an (C₁-C₃)BlCOhOl, or an (C₂-C₃)ester.

13. The use according to claim 12, wherein Y is O^"Cat+ or a nucleoside; R1 is a methyl; A is O or CH₂; n is 2; X is a bromide; B is O; and m is 1 or 2.

14. The use according to claim 12, wherein said phosphoantigen compound comprises the compounds of formula (III) or (IV) :

(III)

in which X is a halogen, m is an integer from 1 to 3, R1 is a methyl or ethyl group, Cat+ represents at least one identical or different organic or mineral cation(s) or protons, n is an integer from 2 to 20, and Y is O^'Cat÷, a nucleoside, or a radical -A-R₀, wherein R₀ is selected from the group of:

1)

OH

(CH₂)_n C R₂

Ri wherein n is an integer from 2 to 20, R₁ is a (CVC^alkyl group, and R₂ is an halogenated

(Ci-C₃)alkyl, a

an halogenated (C₂-C₃)acyl or a (C-i-C₃)alkoxy- (C₂-C₃)acyl;

2)

wherein n is an integer from 2 to 20, and R₁ is a methyl or ethyl group; and

3)

R₃ ,R₅

— C — W

R₄ \ wherein R₃, R₄, and R₅ , identical or different, are a hydrogen or (CrC₃)alkyl group, W is CH- or -N-, and R₆ is an (C₂-C₃)acyl, an aldehyde, an (CrC₃)alcohol, or an (C₂-C₃)ester.

15. The use according to claim 12, wherein said phosphoantigen compound comprises formula (V):

16. The use according to claim 12, wherein said phosphoantigen compound comprises formula (Vl):

and x Cat+ is one or two sodium atoms (Na⁺).

17. The use according to claim 12, wherein said phosphoantigen compound comprises formula (VII):

and x Cat+ is one or two sodium atoms (Na⁺).

18. The use according to claim 1 , wherein said phosphoantigen compound is selected from the group consisting of:

3-(bromomethyl)-3-butanol-1-yl-diphosphate (BrHPP); 5-bromo-4-hydroxy-4-methylpentyl pyrophosphonate (CBrHPP);

3-(iodomethyI)-3-butanol-1-yl-diphosphate (IHPP);

3-(chloromethyl)-3-butanol-1 -yl-diphosphate (CIHPP);

3-(bromomethyl)-3-butanol-1 -yl-triphosphate (BrHPPP);

3-(iodomethyI)-3-butanol-1 -yl-triphosphate (IHPPP); α,γ-di-[3-(bromomethyl)-3-butanol-1-yl]-triphosphate (diBrHTP); or α,γ-di-[3-(iodomethyl)-3-butanol-1-yl]-triphosphate (dilHTP).