WO2008046576A1

WO2008046576A1 - Use of glucocorticoid antagonists in vaccination

Info

Publication number: WO2008046576A1
Application number: PCT/EP2007/008936
Authority: WO
Inventors: Jean-Marc Limacher
Original assignee: Transgene S.A.
Priority date: 2006-10-16
Filing date: 2007-10-15
Publication date: 2008-04-24
Also published as: WO2008046576B1

Abstract

The present invention concerns new immunogenic combinations. In particular the present invention provides combination products that comprise recombinant vectors and/or antigen and specific compounds able to improve the antigen related immune response raised in vivo by said recombinant vectors.

Description

USE OF GLUCOCORTICOID ANTAGONISTS IN VACCINATION

The present invention relates to methods and compositions for improving the immune response raised in vivo by an immunogenic composition, in particular a vaccine. In particular the present invention provides methods and compositions for improving the said immune response by regulating the blood levels of corticosteroids, and more specifically of glucocorticoids.

Traditional vaccination techniques involving the introduction into an animal system of an antigen (e.g. peptides, proteins) which can induce an immune response, and thereby protect said animal against infection for example, have been known for many years. These techniques have further included the development of both live and inactivated vaccines. Live vaccines are typically attenuated nonpathogenic versions of an infectious agent that are capable of priming an immune response directed against a pathogenic version of the infectious agent.

In recent years there have been advances in the development of recombinant vaccines, especially recombinant live vaccines, in which foreign antigens of interest are encoded and expressed from a vector. Amongst them, vectors based on recombinant viruses have shown great promise and play an important role in the development of new vaccines. Many viruses have been investigated for their ability to express proteins from foreign pathogens or tumoral tissue, and to induce specific immunological responses against these antigens in vivo. Generally, these gene-based vaccines can stimulate potent humoral and cellular immune responses and viral vectors might be an effective strategy for both the delivery of antigen-encoding genes and the facilitation and enhancement of antigen presentation. In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen-specific antigens to the immune system. Furthermore, the vector system must meet criteria that enable its production on a large-scale basis. Several viral vaccine vectors have thus emerged to date, all of them having relative advantages and limits depending on the proposed application (for a review on recombinant viral vaccines see for example Harrop and Carroll, 2006, Front Biosci., 11, 804-817 ; Yokoyama et al . , 1997, J Vet Med Sci.,59, 311-322) .

Following the observation in the early 1990 's that plasmid DNA vectors could directly transfect animal cells in vivo, significant research efforts have also been undertaken to develop vaccination techniques based upon the use of DNA plasmids to induce immune response, by direct introduction into animals of DNA which encodes for antigens. Such techniques which are widely referred as DNA vaccination have now been used to elicit protective immune responses in large number of disease models. For a review on DNA vaccines, see Reyes-Sandoval and Ertl, 2001 (Current Molecular Medicine, 1, 217-243) .

Besides, there has been a major effort in recent years, with significant success, to discover new drug compounds that act by stimulating certain key aspects of the immune system which will serve to increase the immune response induced by vaccines. Most of these compounds, referred as immune response modifiers (IRMs) or adjuvants, appear to act through basic immune system mechanisms via Toll-like receptors (TLRs) to induce various important cytokines biosynthesis (e.g., interferons, interleukins, tumor necrosis factor, etc.)- Such compounds have been shown to stimulate a rapid release of certain dendritic cell, monocyte/macrophage-derived cytokines and are also capable of stimulating B cells to secrete antibodies which play an important role in the antiviral and antitumor activities of IRM compounds. One of the predominant immunostimulating responses induced by IRMs is the induction of interferon alpha (INF-α) production, which is believed to be very important in the acute antiviral and antitumor activities seen. Moreover, up regulation of other cytokines such as, for example, tumor necrosis factor (TNF) , IL-I and IL-6 also have potentially beneficial activities and are believed to contribute to the antiviral and antitumor properties of these compounds. Immune response modifiers (IRMs) have been disclosed as useful for treating a wide variety of diseases and conditions, including viral diseases (e.g., human papilloma virus, hepatitis, herpes, HIV infection), neoplasias (e.g., basal cell carcinoma, squamous cell carcinoma, actinic keratosis, melanoma), and Th2-mediated diseases (e.g., asthma, allergic rhinitis, atopic dermatitis) .

Examples of such immune response modifiers (IRMs), include the CpG oligonucleotides (see US 6,194,388; US2006094683; WO 2004039829 for examples), lipopolysaccharides, polyinosicipolycytidylic acid complexes (Kadowaki, et al., 2001, J. Immunol. 166, 2291-2295), and polypeptides and proteins known to induce cytokine production from dendritic cells and/or monocyte/macrophages. Other examples of such immune response modifiers (IRMs) are small organic molecules such as imidazoquinolinamines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, imidazonaphthyridine amines, oxazoloquinoline amines, thiazoloquinoline amines and 1,2-bridged imidazoquinoline amines (see for example US 4,689,338; US 5,389,640; US 6,110,929; and US 6,331,539).

In its attempts to develop new immune response modifiers which will serve to increase the immune response induced by vaccines, the Applicant has now surprisingly found that glucocorticoid antagonists can be used in methods for eliciting an enhanced immune response to at least one antigen in a patient.

Corticosteroids are steroid hormones structurally related to cholesterol. These hormones are synthesized in the adrenal cortex and include the glucocorticoids (e.g. Cortisol), the mineralocorticoids (e.g aldosterone) as well as weak androgens and estrogens. The adrenal function, like that of the thyroid gland, is under the control of the hypothalamus (HPT) and the pituitary gland (PIT) . When Cortisol (the naturally-occurring glucocorticoid) levels drop below a set point, the hypothalamus releases CRH (corticotropin releasing hormone) which stimulates adrenocorticotropic hormone (ACTH) release from the pituitary gland. ACTH is a tropic hormone which stimulates the synthesis and secretion of Cortisol (it has minimal effects on aldosterone synthesis/secretion) , and the growth of the adrenal gland. When Cortisol levels increase, this shuts off CRH and ACTH secretion. Cortisol is characterized by its properties related to the biosynthesis and metabolism of glucose; this is the reason why Cortisol and natural or synthetic analogues thereof are usually named glucocorticoids. They bind to the glucocorticoid receptor (GR) . Additionally, glucocorticoids regulate a wide variety of immune cell functions and expression of immune molecules. More specifically, glucocorticoids modulate the transcription of many cytokines. They suppress the proinflammatory cytokines IL-I, IL-2, IL-6, IL-8, IL-Il, IL-12, TNF-α, INF-γ and GM-CSF while upregulating the anti-inflammatory cytokines IL-4 and IL-IO (for a review see for example Webster et al, 2002, Annu. Rev. Immunol., 20, 125-163).

Numerous glucocorticoid antagonists are known in the art and have been developed for treatment of hypercortisolaemia and related disorders, such as for example Cushing' s syndrome (Newell-Price et al., 2006, Lancet, ^• 367, 1605-1617), depression (Wolkowitz and Reus, 1999, Psychosomatic Medicine, 61, 698-711), heart failure (US 6,881,739), or postpartum psychosis (US 2004/0229855) .

The present invention relates to the use of glucocorticoid antagonists for the preparation of immunogenic combination wherein said glucocorticoid antagonists elicit an enhanced immune response to at least one antigen administered to a patient.

The present invention further relates to methods for eliciting an enhanced immune response to at least one antigen in a patient, comprising administering to the patient, either sequentially or simultaneously, a safe and effective amount of (i) an immunogenic composition and (ii) at least one g -3lucocorticoid antagonist.

According to special embodiment, the present invention relates to the use of glucocorticoid antagonists for the preparation of immunogenic combination wherein said glucocorticoid antagonists elicit an enhanced immune response to at least one antigen administered to a patient, wherein said antigen binds to MHC class II molecules (and is recognized by CD4+ T cells) .

The present invention further relates to methods for eliciting an enhanced immune response to at least one antigen in a patient, comprising administering to the patient, either sequentially or simultaneously, a safe and effective amount of (i) an immunogenic composition and (ii) at least one glucocorticoid antagonist and wherein said antigen binds to MHC class II molecules (and is recognized by CD4+ T cells) .

The present invention relates to an improvement to vaccines, more particularly to recombinant vaccines expressing in vivo at least one heterologous nucleotide sequence, especially a nucleotide sequence encoding an antigen. It relates in particular to a combination of an immunogenic composition, in particular a recombinant vaccine expressing at least one antigen, with at least one glucocorticoid antagonist which is capable of increasing in vivo the immune response raised against the said immunogenic composition relative to the same composition with no glucocorticoid antagonists (i.e. "an enhanced immune response") . According to one specific embodiment, the said immune response is a CD4+ T cell response.

As used herein throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced compounds or steps, unless the context dictates otherwise. For example, the term "a cell" includes a plurality of cells including a mixture thereof. More specifically, "at least one" and "one or more" means a number which is one or greater than one, with a special preference for one, two or three.

The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

The term -"about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

As used herein, the term "comprising", "containing" when used to define products, compositions and methods, is intended to mean that the products, compositions and methods include the referenced compounds or steps, but not excluding others.

The term "patient" refers to a vertebrate, particularly a member of the mammalian species and includes, but is not limited to, domestic animals, sport animals, primates including humans. The term "patient" is in no way limited to a special disease status, it encompasses both patients who have already developed a disease of interest and patients who are not sick.

As used herein, the term "treatment" or "treating" encompasses prophylaxis and/or therapy. Accordingly the immunogenic combinations or methods of the present invention are not limited to therapeutic applications and can be used in prophylaxis ones. "Prophylaxis" is not limited to preventing immediate diseases (e.g. infectious diseases), it further encompasses prevention of long term consequences of these infections such as cirrhosis or cancer.

The subject matter of the present invention is therefore an immunogenic combination containing at least one glucocorticoid antagonist.

According to another embodiment, the present invention concerns an immunogenic composition the administration of which is combined to the administration to the patient of a safe and effective amount of at least one glucocorticoid antagonist.

As used herein, the terms "immunogenic composition" "vaccine composition", "vaccine" or similar terms can be used interchangeably and mean an agent suitable for stimulating a subject's immune system to ameliorate a current condition or to protect against or to reduce present or future harm or infections (including viral, bacterial, parasitic infections), e.g., reduced tumour cell proliferation or survival, reduced pathogen replication or spread in a subject or a detectably reduced unwanted symptom(s) associated with a condition. Vaccines may modulate, typically detectably enhance, humoral, cell mediated or innate immune responses. Said immunogenic composition can contain at least one antigen and/or at least one recombinant vector expressing in vivo at least one heterologous nucleotide sequence, especially an heterologous nucleotide sequence encoding at least one antigen.

Therefore the Invention concerns a composition product which contains (i) at least one antigen and/or at least one recombinant vector expressing in vivo at least one heterologous nucleotide sequence, especially an heterologous nucleotide sequence encoding at least one antigen and (ii) at least one glucocorticoid antagonist.

As used herein, the term "antigen" refers to any substance that is capable of being the target of an immune response. An antigen may be the target of, for example, a cell-mediated and/or humoral immune response raised by a patient. The term "antigen" encompasses for example all or part of viral antigens, tumour-specific or -related antigens, bacterial antigens, parasitic antigens, allergens and the like:

Viral antigens include for example antigens from hepatitis viruses A, B, C, D and E, HIV, herpes viruses, cytomegalovirus, varicella zoster, papilloma viruses, Epstein Barr virus, influenza viruses, para-influenza viruses, adenoviruses, coxsakie viruses, picorna viruses, rotaviruses, respiratory syncytial viruses, pox viruses, rhinoviruses, rubella virus, papovirus, mumps virus, measles virus; some non-limiting examples of known viral antigens include the following : antigens derived from HIV-I such as tat, nef, gpl20 or gplβO, gp40, p24, gag, env, vif, vpr, vpu, rev or part and/or combinations thereof; antigens derived from human herpes viruses such as gH, gL gM gB gC gK gE or gD or part and/or combinations thereof or Immediate Early protein such asICP27, ICP47, ICP4, ICP36 from HSVl or HSV2 ; antigens derived from cytomegalovirus, especially human cytomegalovirus such as gB or derivatives thereof ; antigens derived from Epstein Barr virus such as gp350 or derivatives thereof; antigens derived from Varicella Zoster Virus such as gpl, 11, 111 and IE63; antigens derived from a hepatitis virus such as hepatitis B , hepatitis C or hepatitis E virus antigen (e.g. env protein El or E2, core protein, NS2, NS3, NS4a, NS4b, NS5a, NS5b, p7, or part and/or combinations thereof of HCV) ; antigens derived from human papilloma viruses (for example HPV6, 11, 16,18, e.g. Ll, L2, El, E2, E3, E4, E5, E6, E7, or part and/or combinations thereof) ; antigens derived from other viral pathogens, such as Respiratory Syncytial virus (e.g. F and G proteins or derivatives thereof), parainfluenza virus, measles virus, mumps virus, flaviviruses (e. g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus, Japanese Encephalitis Virus) or Influenza virus cells (e.g. HA, NP, NA, or M proteins, or part and/or combinations thereof) ; tumor-specific or -related antigens include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, renal cancer, malignant melanoma, laryngeal cancer, prostate cancer. Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53) , fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Some non-limiting examples of tumor-specific or -related antigens include MART- 1/Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV) , adenosine deaminase-binding protein (ADAbp) , cyclophilin b, Colorectal associated antigen (CRC) -C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-I and CAP-2, etvβ, amll, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-I, PSA-2, and PSA-3, prostate- specific membrane antigen (PSMA) , T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-Aβ, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-AlO, MAGE-AIl, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-Cl, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE- 1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-I, NAG, GnT-V, MUM-I, CDK4, tyrosinase, p53, MUC family (e.g. MUC-I), HER2/neu, p21ras, RCASl, alpha-fetoprotein, E-cadherin, alpha-catenin, beta- catenin and gamma-catenin, pl20ctn, gplOO . sup. Pmelll7, PRAME, NY-ESO-I, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, pl5, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, litιp-1, PlA, EBV- encoded nuclear antigen (EBNA)-I, brain glycogen phosphorylase, SSX-I, SSX-2 (HOM-MEL-40) , SSX-I, SSX-4, SSX-5, SCP-I and CT-7, and c-erbB-2;

- bacterial antigens includes for example antigens from Mycobacteria causing TB and leprosy, pneumocci, aerobic gram negative bacilli, mycoplasma, staphyloccocal infections, streptococcal infections, salmonellae, chlamydiae, neisseriae; other antigens includes for example antigens from malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis, filariasis; allergens refer to a substance that can induce an allergic or asthmatic response in a susceptible subject. The list of allergens is enormous and can include pollens, insect venoms, animal dander dust, fungal spores and drugs (e.g. penicillin) . Examples of natural, animal and plant allergens include but are not limited to proteins specific to the following genuses: Canine (Canis familiaris) ; Dermatophagoides (e.g. Dermatophagoides farinae) ; Felis (Felis domesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum) ; Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata) ; Alder; Alnus (Alnus gultinoasa) ; Betula (Betula verrucosa) ; Quercus (Quercus alba) ; Olea (Olea europa) ; Artemisia (Artemisia vulgaris) ; Plantago (e.g. Plantago lanceolate); Parietaria (e.g. Parietaria officinalis or Parietaria judaica) ; Blattella (e.g. Blattella germanica) ; Apis (e.g. Apis multiflorum) ; Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa) ; Juniperus (e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei); Thuya (e.g. Thuya orientalis) ; Chamaecyparis (e.g.

Chamaecyparis obtusa) ; Periplaneta (e.g. Periplaneta americana) ; Agropyron (e.g. Agropyron repens) ; Secale (e.g.

Secale cereale) ; Triticum (e.g. Triticum aestivum) ; Dactylis

(e.g. Dactylis glomerata) ; Festuca (e.g. Festuca elatior) ; Poa

(e.g. Poa pratensis or Poa compressa) ; Avena (e.g. Avena sativa) ; Holcus (e.g. Holcus lanatus) ; Anthoxanthum (e.g.

Anthoxanthum odoratum) ; Arrhenatherum (e.g. Arrhenatherum elatius) ; Agrostis (e.g. Agrostis alba); Phleum (e.g. Phleum pratense) ; Phalaris (e.g. Phalaris arundinacea) ; Paspalum

(e.g. Paspalum notatum) ; Sorghum (e.g. Sorghum halepensis) ; and Bromus (e.g. Bromus inermis).

The term antigen further concerns complex antigen, such as live vaccines which are typically attenuated non-pathogenic versions of an infectious agent.

According to one special embodiment, said antigen binds to MHC class II molecules (and is recognized by CD4+ T cells) . According to the invention, these CD4+ T cell antigens are at least 10 amino acids, more usually at least 13 amino acids in length, and can be much longer.

According to one special embodiment, said antigen is encoded by an heterologous nucleotide sequence and is expressed in vivo by a recombinant vector.

In a particularly preferred embodiment the heterologous nucleotide sequence of the present invention, encodes one or more of all or part of the following antigens HBV-PreSl PreS2 and Surface env proteins, core and polHIV- gpl20 gp40,gpl60, p24, gag, pol, env, vif, vpr, vpu, tat, rev, nef; HPV-El, E2, E3, E4, E5, E6, E7, E8, Ll, L2 (see for example WO 90/10459, WO 98/04705, WO 99/03885); HCV env protein El or E2, core protein, NS2, NS3, NS4a, NS4b, NS5a, NS5b, p7 (see for example wo2004lll082 , WO2005051420) ; Muc-1 (see for example US 5 , 861 , 381 ; US6 , 054 , 438 ; WO98 /04727 ;

WO98 /37095 ) .

According to variants of the invention, the composition product contains at least two antigens, or an heterologous nucleotide sequence encoding at least two antigens, or at least two heterologous nucleotide sequences encoding at least two antigens, or any combination thereof.

In special embodiment, the said composition product comprises a source of one or more CD4+ T cell epitopes of the target antigen (i.e. an epitope which binds to MHC class I molecules and is recognized by CD4+ T cells) .

According to another special embodiment, said heterologous nucleotide sequence of the present invention, encodes all or part of HPV antigen (s) selected in the group consisting of E6 early coding region of HPV, E7 early coding region of HPV and derivates or combination thereof.

The HPV antigen encoded by the recombinant vector according to the invention is selected in the group consisting of an HPV E6 polypeptide, an HPV E7 polypeptide or both an HPV E6 polypeptide and an HPV E7 polypeptide. The present invention encompasses the use of any HPV E6 polypeptide which binding to p53 is altered or at least significantly reduced and/or the use of any HPV E7 polypeptide which binding to Rb is altered or at least significantly reduced (Munger et al., 1989, EMBO J. 8, 4099-4105; Crook et al., 1991, Cell 67, 547- 556; Heck et al., 1992, Proc. Natl. Acad. Sci. USA 89, 4442- 4446; Phelps et al., 1992, J. Virol. 66, 2148-2427). A non- oncogenic HPV-16 E6 variant which is suitable for the purpose of the present invention is deleted of one or more amino acid residues located from approximately position 118 to approximately position 122 (+1 representing the first methionine residue of the native HPV-16 E6 polypeptide), with a special preference for the complete deletion of residues 118 to 122 (CPEEK) . A non-oncogenic HPV-16 E7 variant which is suitable for the purpose of the present invention is deleted of one or more amino acid residues located from approximately position 21 to approximately position 26 (+1 representing the first amino acid of the native HPV-lβ E7 polypeptide, with a special preference for the complete deletion of residues 21 to 26 (DLYCYE) . According to a preferred embodiment, the one or more HPV-16 early polypeptide (s) in use in the invention is/are further modified so as to improve MHC class I and/or MHC class II presentation, and/or to stimulate anti-HPV immunity. HPV E6 and E7 polypeptides are nuclear proteins and it has been previously shown that membrane presentation permits to improve their therapeutic efficacy (see for example WO99/03885) . Thus, it may be advisable to modify at least one of the HPV early polypeptide (s) so as to be anchored to the cell membrane. Membrane anchorage can be easily achieved by incorporating in the HPV early polypeptide a membrane- anchoring sequence and if the native polypeptide lacks it a secretory sequence (i.e. a signal peptide). Membrane-anchoring and secretory sequences are known in the art. Briefly, secretory sequences are present at the N-terminus of the membrane presented or secreted polypeptides and initiate their passage into the endoplasmic reticulum (ER) . They usually comprise 15 to 35 essentially hydrophobic amino acids which are then removed by a specific ER-located endopeptidase to give the mature polypeptide. Membrane-anchoring sequences are usually highly hydrophobic in nature and serves to anchor the polypeptides in the cell membrane (see for example Branden and Tooze, 1991, in Introduction to Protein Structure p. 202-214, NY Garland) .

The choice of the membrane-anchoring and secretory sequences which can be used in the context of the present invention is vast. They may be obtained from any membrane- anchored and/or secreted polypeptide comprising it (e.g. cellular or viral polypeptides) such as the rabies glycoprotein, of the HIV virus envelope glycoprotein or of the measles virus F protein or may be synthetic. The membrane anchoring and/or secretory sequences inserted in each of the early HPV-16 polypeptides used according to the invention may have a common or different origin. The preferred site of insertion of the secretory sequence is the N-terminus downstream of the codon for initiation of translation and that of the membrane-anchoring sequence is the C-terminus, for example immediately upstream of the stop codon.

The HPV E6 polypeptide in use in the present invention is preferably modified by insertion of the secretory and membrane-anchoring signals of the measles F protein. Optionally or in combination, the HPV E7 polypeptide in -use in the present invention is preferably modified by insertion of the secretory and membrane-anchoring signals of the rabies glycoprotein.

The therapeutic efficacy of the recombinant vector can also be improved by using one or more nucleic acid encoding immunopotentiator polypeptide (s) . For example, it may be advantageous to link the HPV early polypeptide (s) to a polypeptide such as calreticulin (Cheng et al., 2001, J. Clin. Invest. 108, 669-678), Mycobacterium tuberculosis heat shock protein 70 (HSP70) (Chen et al., 2000, Cancer Res. 60, 1035- 1042), ubiquitin (Rodriguez et al., 1997, J. Virol. 71, 8497- 8503) or the translocation domain of a bacterial toxin such as Pseudomonas aeruginosa exotoxin A (ETA(dΙII)) (Hung et al., 2001 Cancer Res. 61, 3698-3703).

According to another and preferred embodiment, the recombinant vector according to the invention comprises a nucleic acid encoding one or more early polypeptide (s) as above defined, and more particularly HPV-16 and/or HPV-18 early E6 and/or E7 polypeptides.

According to another special embodiment, said heterologous nucleotide sequence of the present invention, encodes all or part of MUC 1 antigen or derivates thereof.

According to another special embodiment, said heterologous nucleotide sequence of the present invention, encodes one or more of all or part of the followings: HCV env protein El or E2, core protein, NS2, NS3, NS4a, NS4b, NS5a, NS5b, -p7 or derivates thereof. According to another special embodiment, said heterologous nucleotide sequence of the present invention, encodes one or more fusion protein wherein the configuration is not native in the sense that at least one of the NS polypeptides appears in an order which is distinct from that of the native configuration. Thus, if the fusion protein comprises a NS3 polypeptide, a NS4A polypeptide and a NS5B polypeptide, the native configuration would be NS3-NS4A-NS5B with NS3 at the N-terminus and NS5B at the C-terminus . In contrast, a non-native configuration can be NS5B-NS3-NS4A, NS5B-NS4A-NS3, NS4A-NS3-NS5B, NS4A-NS5B-NS3 or NS3-NS5B-NS4A. In particular, the fusion protein according to the invention comprises at least one of the followings: o A NS4A polypeptide fused directly or through a linker to the N-terminus of a NS3 polypeptide; o A NS3 polypeptide fused directly or through a linker to the N-terminus of a NS5B polypeptide; o A NS4B polypeptide fused directly or through a linker to the N-terminus of a NS5B polypeptide; o A NS4A polypeptide fused directly or through a linker to the N-terminus of a NS3 polypeptide which is fused directly or through a linker to the N-terminus of a

NS4B polypeptide; and/or o A NS3 polypeptide fused directly or through a linker to the N-terminus of a NS4B polypeptide which is fused directly or through a linker to the N-terminus of a NS5B polypeptide.

In such specific portions of the fusion protein of the invention, each of the NS polypeptides can be independently native or modified. For example, the NS4A polypeptide included in the NS4A-NS3 portion can be native whereas the NS3 polypeptide comprises at least one of the modifications described below.

If needed, the nucleic acid molecule in use in the invention may be optimized for providing high level expression of the antigen (e.g. HPV early polypeptide (s) ) in a particular host cell or organism, e.g. a human host cell or organism. Typically, codon optimisation is performed by replacing one or more "native" (e.g. HPV) codon corresponding to a codon infrequently used in the mammalian host cell by one or more codon encoding the same amino acid which is more frequently used. This can be achieved by conventional mutagenesis or by chemical synthetic techniques (e.g. resulting in a synthetic nucleic acid) . It is not necessary to replace all native codons corresponding to infrequently used codons since increased expression can be achieved even with partial replacement. Moreover, some deviations from strict adherence to optimised codon usage may be made to accommodate the introduction of restriction site(s).

As used herein, the term "recombinant vector" refers to viral as well as non viral vectors, including extrachromosomal (e.g. episome) , multicopy and integrating vectors (i.e. for being incorporated into the host chromosomes). Particularly important in the context of the invention are vectors for use in gene therapy (i.e. which are capable of delivering the nucleic acid to a host organism) as well as expression vectors for use in various expression systems. Suitable non viral vectors include plasmids such as pREP4, pCEP4 (Invitrogene) , pCI (Promega), pCDM8 (Seed, 1987, Nature 329, 840), pVAX and pgWiz (Gene Therapy System Inc; Himoudi et al., 2002, J. Virol. 76, 12735-12746). Suitable viral vectors may be derived from a variety of different viruses (e.g. retrovirus, adenovirus, AAV, poxvirus, herpes virus, measle virus, foamy virus and the like) . As used herein, the term "viral vector" encompasses vector DNA/RNA as well as viral particles generated thereof. Viral vectors can be replication-competent, or can be genetically disabled so as to be replication- defective or replication-impaired. The term "replication- competent" as used herein encompasses replication-selective and conditionally-replicative viral vectors which are engineered to replicate better or selectively in specific host cells (e.g. tumoral cells).

In one aspect, the recombinant vector in use in the invention is a recombinant adenoviral vector (for a review, see "Adenoviral vectors for gene therapy", 2002, Ed D. Curiel and J. Douglas, Academic Press) . It can be derived from a variety of human or animal sources and any serotype can be employed from the adenovirus serotypes 1 through 51. Particularly preferred are human adenoviruses 2 (Ad2), 5 (Ad5), 6 (Ad6), 11 (AdIl), 24 (Ad24) and 35 (Ad35). Such adenovirus are available from the American Type Culture Collection (ATCC, Rockville, Md.), and have been the subject of numerous publications describing their sequence, organization and methods of producing, allowing the artisan to apply them (see for example US 6,133,028; US 6,110,735; WO 02/40665; WO 00/50573; EP 1016711; Vogels et al., 2003, J. Virol. 77, 8263-8271) .

The adenoviral vector in use in the present invention can be replication-competent. Numerous examples of replication- competent adenoviral vectors are readily available to those skill in the art (see, for example, Hernandez-Alcoceba et al., 2000, Human Gene Ther. 11, 2009-2024; Nemunaitis et al., 2001, Gene Ther. 8, 746-759; Alemany et al., 2000, Nature Biotechnology 18, 723-727) . For example, they can be engineered from a wild-type adenovirus genome by deletion in the ElA CR2 domain (see for example WO00/24408) and/or by replacement of the native El and/or E4 promoters with tissue, tumor or cell status-specific promoters (see for example US 5,998,205, WO99/25860, US5, 698,443, WO00/46355, WO00/15820 and WO01/36650) .

Alternatively, the adenoviral vector in use in the invention is replication-defective (see for example WO94/28152; Lusky et al., 1998, J. Virol 72, 2022-2032). Preferred replication-defective adenoviral vectors are El- defective (see for example US 6,136,594 and US 6,013,638), with an El deletion extending from approximately positions 459 to 3328 or from approximately positions 459 to 3510 (by reference to the sequence of the human adenovirus type 5 disclosed in the GeneBank under the accession number M 73260 and in Chroboczek et al., 1992, Virol. 186, 280-285). The cloning capacity can further be improved by deleting additional portion (s) of the adenoviral genome (all or part of the non essential E3 region or of other essential E2, E4 regions) . Insertion of a nucleic acid in any location of the adenoviral vector can be performed through homologous recombination as described in Chartier et al. (1996, J. Virol. 70, 4805-4810) . For example, the nucleic acid encoding the HPV-16 E6 polypeptide can be inserted in replacement of the El region and the nucleic acid encoding the HPV-16 E7 polypeptide in replacement of the E3 region or vice versa.

In another and preferred aspect, the vector in use in the invention is a poxviral vector (see for example Cox et al. in "Viruses in Human Gene Therapy" Ed J. M. Hos, Carolina Academic Press) . According to another preferred embodiment it is selected in the group consisting of vaccinia virus, suitable vaccinia viruses include without limitation the Copenhagen strain (Goebel et al., 1990, Virol. 179, 247-266 and 517-563; Johnson et al., 1993, Virol. 196, 381-401), the Wyeth strain and the highly attenuated attenuated virus derived thereof including MVA (for review see Mayr, A., et al., 1975, Infection 3, 6-14) and derivates thereof (such as MVA vaccinia strain 575 (ECACC V00120707 - US 6,913,752), NYVAC (see WO 92/15672 - Tartaglia et al., 1992, Virology, 188, 217-232) . Determination of the complete sequence of the MVA genome and comparison with the Copenhagen W genome has allowed the precise identification of the seven deletions (I to VII) which occurred in the MVA genome (Antoine et al., 1998, Virology 244, 365-396), any of which can be used to insert the antigen-encoding nucleic acid. The vector may also be obtained from any other member of the poxviridae, in particular fowlpox (e.g. TROVAC, see Paoletti et al, 1995, Dev Biol Stand., 84, 159-163); canarypox (e.g. ALVAC, WO 95/27780, Paoletti et al, 1995, Dev Biol Stand., 84, 159-163); pigeonpox; swinepox and the like. By way of example, persons skilled in the art may refer to WO 92 15672 (incorporated by reference) which describes the production of expression vectors based on poxviruses capable of expressing such heterologous nucleotide sequence, especially nucleotide sequence encoding antigen.

The basic technique for inserting the nucleic acid and associated regulatory elements required for expression in a poxviral genome is described in numerous documents accessible to the man skilled in the art (Paul et al., 2002, Cancer gene Ther. 9, 470-477; Piccini et al., 1987, Methods of Enzymology 153, 545-563 ; US 4,769,330 ; US 4,772,848 ; US 4,603,112 ; US 5,100,587 and US 5,179,993). Usually, one proceed through homologous recombination between overlapping sequences (i.e. desired insertion site) present both in the viral genome and a plasmid carrying the nucleic acid to insert.

The nucleic acid encoding the antigen of the Invention is preferably inserted in a nonessential locus of the poxviral genome, in order that the recombinant poxvirus remains viable and infectious. Nonessential regions are non-coding intergenic regions or any gene for which inactivation or deletion does not significantly impair viral growth, replication or infection. One may also envisage insertion in an essential viral locus provided that the defective function is supplied in trans during production of viral particles, for example by using an helper cell line carrying the complementing sequences corresponding to those deleted in the poxviral genome.

When using the Copenhagen vaccinia virus, the antigen- encoding nucleic acid is preferably inserted in the thymidine kinase gene (tk) (Hruby et al., 1983, Proc. Natl. Acad. Sci USA 80, 3411-3415 ; Weir et al., 1983, J. Virol. 46, 530-537). However, other insertion sites are also appropriate, e.g. in the hemagglutinin gene (Guo et al., 1989, J. Virol. 63, 4189- 4198), in the KlL locus, in the u gene (Zhou et al., 1990, J. Gen. Virol. 71, 2185-2190) or at the left end of the vaccinia virus genome where a variety of spontaneous or engineered deletions have been reported in the literature (Altenburger et al., 1989, Archives Virol. 105, 15-27 ; Moss et al. 1981, J. Virol. 40, 387-395 ; Panicali et al., 1981, J. Virol. 37, 1000-1010 ; Perkus et al, 1989, J. Virol. 63, 3829-3836 ; Perkus et al, 1990, Virol. 179, 276-286 ; Perkus et al, 1991, Virol. 180, 406-410) .

When using MVA, the antigen-encoding nucleic acid can be inserted in anyone of the identified deletions I to VII as well as in the D4R locus, but insertion in deletion II or III is preferred (Meyer et al., 1991, J. Gen. Virol. 72, 1031-1038 ; Sutter et al . , 1994, Vaccine 12, 1032-1040).

When using fowlpox virus, although insertion within the thymidine kinase gene may be considered, the antigen-encoding nucleic acid is preferably introduced in the intergenic region situated between ORFs 7 and 9 (see for example EP 314 569 and US 5,180, 675) .

Preferably, the antigen-encoding nucleic acid in use in the invention is in a form suitable for its expression in a host cell or organism, which means that the nucleic acid sequence encoding the antigen are placed under the control of one or more regulatory sequences necessary for its expression in the host cell or organism. As used herein, the term "regulatory sequence" refers to any sequence that allows, contributes or modulates the expression of a nucleic acid in a given host cell, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (i.e. mRNA) into the host cell. It will be appreciated by those skilled in the art that the choice of the regulatory sequences can depend on factors such as the host cell, the vector and the level of expression desired. The nucleic acid encoding the antigen is operatively linked to a gene expression sequence which directs the expression of the antigen nucleic acid within a eukaryotic cell. The gene expression sequence is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the antigen nucleic acid to which it is operatively linked. The gene expression sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT) , adenosine deaminase, pyruvate kinase, b-actin promoter and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV) , Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art. In general, the gene expression sequence shall include, as necessary, 5' non-transcribing and 5' non- translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5¹ non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined antigen nucleic acid. The gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired. Preferred promoters for use in a poxviral vector (see below) include without limitation vaccinia promoters 7.5K, H5R, TK, p28, pll and KlL, chimeric promoters between early and late poxviral promoters as well as synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechniques 23, 1094-1097), Hammond et al. (1997, J. Virological Methods 66, 135-138) and Kumar and Boyle (1990, Virology 179, 151-158) .

The promoter is of special importance and the present invention encompasses the use of constitutive promoters which direct expression of the nucleic acid in many types of host cell and those which direct expression only in certain host cells or in response to specific events or exogenous factors (e.g. by temperature, nutrient additive, hormone or other ligand) . Suitable promoters are widely described in literature and one may cite more specifically viral promoters such as RSV, SV40, CMV and MLP promoters. Preferred promoters for use in a poxviral vector include without limitation vaccinia promoters 7.5K, H5R, TK, p28, pll and KlL, chimeric promoters between early and late poxviral promoters as well as synthetic promoters such as those described in Chakrabarti et al. (1997, Biotechniques 23, 1094-1097), Hammond et al. (i997, J. Virological Methods 66, 135-138) and Kumar and Boyle (1990, Virology 179, 151-158) .

Those skilled in the art will appreciate that the regulatory elements controlling the expression of the nucleic acid molecule of the invention may further comprise additional elements for proper initiation, regulation and/or termination of transcription (e.g. polyA transcription termination sequences), mRNA transport (e.g. nuclear localization signal sequences), processing (e.g. splicing signals), and stability (e.g. introns and non-coding 5' and 3' sequences), translation (e.g. peptide signal, propeptide, tripartite leader sequences, ribosome binding sites, Shine-Dalgamo sequences, etc.) into the host cell or organism.

Alternatively, the recombinant vector in use in the present invention can further comprise at least one nucleic acid encoding at least one cytokine. Suitable cytokines include without limitation interleukins (e.g. IL-2, IL-7, IL- 15, IL-18, IL-21) and interferons (e.g. IFNγ, INFα) , with a special preference for interleukin IL-2. When the recombinant vaccine of the invention comprises a cytokine-expressing nucleic acid, said nucleic acid may be carried by the recombinant vector encoding the one or more antigen (s) or by an independent recombinant vector which can be of the same or a different origin.

Infectious viral particles comprising the above-described recombinant viral vector can be produced by routine process. An exemplary process comprises the steps of: a. introducing the viral vector into a suitable cell line, b. culturing said cell line under suitable conditions so as to allow the production of said infectious viral particle, c. recovering the produced infectious viral particle from the culture of said cell line, and d. optionally purifying said recovered infectious viral particle.

Cells appropriate for propagating adenoviral vectors are for example 293 cells, PERC6 cells, HER96 cells, or cells as disclosed in WO 94/28152, WO 97/00326, US 6,127,175.

Cells appropriate for propagating poxvirus vectors are avian cells, and most preferably primary chicken embryo fibroblasts (CEF) prepared from chicken embryos obtained from fertilized eggs.

The infectious viral particles may be recovered from the culture supernatant or from the cells after lysis (e.g. by chemical means, freezing/thawing, osmotic shock, mecanic shock, sonication and the like) . The viral particles can be isolated by consecutive rounds of plaque purification and then purified using the techniques of the art (chromatographic methods, ultracentrifugation on caesium chloride or sucrose gradient) .

The subject of the present invention is therefore an composition product containing (i) at least one antigen, and (ii) at least one glucocorticoid antagonist.

The subject of the present invention is furthermore an composition product containing (i) at least one recombinant vector expressing in vivo at least one heterologous nucleotide sequence, especially an heterologous nucleotide sequence encoding at least one antigen, and (ii) at least one glucocorticoid antagonist.

According to one special embodiment, said recombinant vector is a recombinant plasmid DNA or a recombinant viral vector.

According to another special embodiment, said recombinant viral vector is a recombinant adenoviral vector.

According to another special embodiment, said recombinant viral vector is a recombinant vaccinia vector.

According to another special embodiment, said recombinant vaccinia vector is a recombinant MVA vector.

As used herein, the terms "glucocorticoid antagonist", "Cortisol antagonist" or similar terms can be used interchangeably and mean any compound or agent which reduces production and/or release of glucocorticoid or circulating levels of biologically active glucocorticoid or which limits the biological effects of glucocorticoid by inhibiting glucocorticoid receptors competitively or non-competitively, or in any other way. The term includes agents which interfere with the regulation of glucocorticoid synthesis along the so- called hypothalmic-pituitary-adrenal gland (HRA) axis. Thus a "glucocorticoid antagonist" may broadly be regarded as any compound or agent which antagonises or inhibits (i.e. reduces or prevents) glucocorticoid activity. A large number of glucocorticoid antagonist are known in literature, especially in papers concerning treatment of hypercortisolaemia related disorders such as Cushing's syndrome or major depression.

"Glucocorticoid antagonist" includes those compounds or agents which inhibit the synthesis of Cortisol, either by- reducing the production of Cortisol in any form or which cause the production of a modified form of Cortisol which is less biologically active than native, naturally occurring Cortisol. Preferably, Cortisol synthesis inhibitors will act on the Cortisol synthetic pathway in a way which does not significantly affect the normal production of the other steroid hormones, in particular which does not significantly affect production of mineralocorticoids such as aldosterone.

"Glucocorticoid antagonist" further includes those compounds or agents which act via Cortisol (glucocorticoid) receptors .

According to one embodiment. the glucocorticoid antagonist of the Invention is a compound or agent able to reduce, and preferably to inhibit, the ACTH release by the hypophysis. Non limiting examples are a corticotropin- releasing factor (CRF) receptor antagonist (Rivier et al., 1984, Science, 224, 889; Menzaghi Fet al.,1994, J. Pharmacol. Exp. Ther., 269, 564; Dunn and Berridge , 1990, Brain Research Reviews, 15, 71); cyproheptadine (also named Periactine - Krieger and Luria, 1976, N Engl J Med. 43, 1179); desmethylcyproheptadine (Lamberts, 1983, Journal of Endocrinology, 96, 395-400) ; bromocriptine (also named Parlodel - Lamberts et al., 1980, J Clin Endocrinol Metab., 51,307); valproic acid (2-propylpentanoic acid, 2- propylvaleric acid, di-n-propylacetic acid) (used here to refer to valproic acid, sodium valproate and divalproex sodium) (Aggernaes et al., 1988, Acta Psychiatr. Scand., 77, 170-174); and derivatives, analogues, solvates or salts thereof .

According to another embodiment, the glucocorticoid antagonist of the Invention is a compound or agent able to reduce, and preferably to inhibit, corticosteroids synthesis, including compound or agent able to reduce, and preferably to inhibit, activity of enzymes involved in corticosteroids synthesis. -Non limiting examples are 1- (o-chlorophenyl) -1- (p- chlorophenyl) -2, 2-dichloroethane (o,p'-DDD) (also named Lysodren or Mitotane) ; 1- (o-chlorophenyl) -1- (p-chlorophenyl) - 2, 2-dichloropropane (also named Mitometh) ; aminogluthetimide (also named Orimetene) ; ketoconazole (also named Nizoral) and its derivatives, especially Cis-2S,4R and cis- 2R,- 4S isomers (Janssen) ; econazole (Squibb); miconazole (Janssen) ; metyrapone (also named metopyrone) ; trilostane and its derivatives (2 alpha-cyano-4alpha, 5alpha-epoxyandrostan- 17beta-ol-3-one ^' (US 3296295, US2004/0204392) ; derivatives of diphenylmethane (e.g. amphenon B); derivatives of pyridine (e.g . metirapon) ; substituted alpha alpha glutaramide (e.g. aminoglutethimide) ; steroid of the spironolactone family; synthetic steroid ZK91587 ; etomidate ; and derivatives, analogues, solvates or salts thereof. For example, isomers of ketoconazole are known and may be used, individually or in combination (Rotstein et al., J. Med. Chem. (1992) 35, 2818- 2825). The Cis-2S,4R and Cis-2R,4S isomers are particularly preferred for use in accordance with the present invention. These isomers may be used individually or in combination as in the commercially available product Fungoral ( Janssen-Cilag) .

According to another embodiment, the glucocorticoid antagonist of the Invention is a cortisol-sequestering agent. Non limiting examples are cyclodextrins; and derivatives, analogues, solvates or salts thereof (e.g. 6-per-deoxy-6-per- (2, 3-dihydroxypropylthio) -γ-cyclodextrin (see further compounds disclosed in US2004/0048830 or EP1333842, the content of which are incorporated herein by reference) ; and derivatives, analogues, solvates or salts thereof.

According to another embodiment, the glucocorticoid antagonist of the Invention is a compound or agent which limits the biological effects of glucocorticoid by inhibiting glucocorticoid receptors. Non limiting examples are 17 β- hydroxy-llβ- (4-dimethylaminophenyl) -17α- (1-propynyl) estra-4, 9- dien-3-one (also named mifepristone or RU486 or RU 38486 - Roussel-Uclaf) ; non steroid substance (e.g. drotaverina hydrochloride (derive of isoquinoline) or acetylsalicic acid ; 21-hydroxy 6,19 oxidoprogesterone (EP 903146) ; compounds disclosed in US 20050222104 (the content of which is incorporated herein by reference) ; and derivatives, analogues, solvates or salts thereof.

According to another embodiment, the glucocorticoid antagonist of the Invention is a compound or agent selected in^" the group consisting in enkephalin analogue [D-Ala2, MePhe4, Met(o)-ol] enkephalin (DAMME) (Stubbs et al . , 1978, The Lancet , 1225-1227); loperamide (commercially available under the trademark IMODIUM from Janssen Pharmaceutica ) ; dexamethasone ; alprazolam; clonidine (Slowinska-Srzednicka, et al., 1988, European Journal of Clinical Pharmacology, 35 115-121) ; oxytocin (Legros al., 1987, Endocrinologica, 114, 345-349); 6- Mercaptonicotinic (US 4,521,425), and derivatives, analogues, solvates or salts thereof.

"Derivatives, analogues, solvates or salts thereof" encompasses compounds which are structurally related to the primary compound (e.g. ketoconazole) but are functionally equivalent or superior. Thus, a derivative might have a slightly inferior therapeutic activity according to the Invention but is a useful molecule because it exhibits reduced toxicity, is more convenient to formulate or administer etc. Derivatives may include salts or other variants which have been more significantly modified while retaining functionally important structural motifs in common with the primary compound.

According to one embodiment, the glucocorticoid antagonist concentration (e.g. in the composition product or used in combination with the composition product) will be from about 0.0001% to about 10% (unless otherwise indicated, all percentages provided herein are weight/weight with respect to the total formulation), from about 0.01% to about 2%, more particularly from about 0.06 to about 1%, preferably from about 0.1 to about 0.6%.

According to another embodiment, the appropriate dosage of antigen or recombinant vector can be adapted as a function of various parameters, in particular the mode of administration; the composition employed; the age, health, and weight of the host organism; the nature and extent of symptoms; kind of concurrent treatment; the frequency of treatment; and/or the need for prevention or therapy. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by a practitioner, in the light of the relevant circumstances. For general guidance, suitable dosage for a MVA-containing composition varies from about 10⁴ to 10¹⁰ pfu (plaque forming units) , desirably from about 10⁵ and 10⁸ pfu whereas adenovirus-comprising composition varies from about 10⁵ to 10¹³ iu (infectious units), desirably from about 10⁷ and 10¹² iu. A composition based on vector plasmids may be administered in doses of between 10 μg and 20 mg, advantageously between 100 μg and 2 mg. Preferably, the composition is administered at dose(s) comprising from 5xlO⁵ pfu to 5xlO⁷ pfu of MVA vaccinia vector.

The dosing regimen may depend at least in part on many factors known in the art including but not limited to the nature of the glucocorticoid antagonist and antigen or recombinant vector used, the nature of the carrier, the amount of the glucocorticoid antagonist and recombinant vector (or antigen) being administered, the state of the subject's immune system (e.g., suppressed, compromised, stimulated), and the method of administering the glucocorticoid antagonist and/or of recombinant vector (or antigen) compounds. Accordingly it is not practical to set forth generally the dosing regimen effective for increasing the efficacy of a immunogenic composition for all possible applications. Those of ordinary skill in the art, however, can readily determine an appropriate dosing regimen with due consideration- of such factors. In some embodiments of the invention, the glucocorticoid antagonist and/or recombinant vector and/or antigen compounds may be administered, for example, once to about once daily, although in some embodiments the glucocorticoid antagonist and/or recombinant vector and/or antigen compounds may be administered at a frequency outside this range. In certain embodiments, the glucocorticoid antagonist and/or recombinant vector and/or antigen compounds may be administered from about once per week to about once per day. In one particular embodiment, the glucocorticoid antagonist and/or recombinant vector and/ or antigen compounds are administered once every week. Desirably, the glucocorticoid antagonist and recombinant vector and/or antigen is administered about 1 to about 10 times at weekly intervals. Preferably, recombinant vector and/or antigen, or any composition containing it, is administered 3 times at weekly intervals by intramuscular or subcutaneous route. Preferably, the glucocorticoid antagonist, or any composition containing it, is administered either sequentially or simultaneously to the administration of said recombinant vector and/or antigen and is administered via enteral route.

In a further aspect, the invention provides a method of increasing an immune response to an antigen in a patient, said method comprising administration, either sequentially or simultaneously, of (i) an immunogenic composition and (ii) at least one glucocorticoid antagonist.

In a special aspect, the invention provides a method of increasing an immune response to an antigen in a patient, said method comprising administration, either sequentially or simultaneously, of (i) at least one antigen and (ii) at least one glucocorticoid antagonist.

In a another special aspect, the invention provides a method of increasing an immune response to an antigen in a patient, said method comprising administration, either sequentially or simultaneously, of (i) a recombinant vector expressing in vivo at least one heterologous nucleotide sequence, especially an heterologous nucleotide sequence encoding at least one antigen and (ii) at least one glucocorticoid antagonist.

In another aspect, the invention provides a method of preventing occurrence of and/or of treating cancer in a patient, said method comprising administration, either sequentially or simultaneously, of (i) an immunogenic composition and (ii) at least one glucocorticoid antagonist.

In one special aspect, the invention provides a method of preventing occurrence of and/or of treating cancer in a patient, said method comprising administration, either sequentially or simultaneously, of (i) at least one antigen and (ii) at least one glucocorticoid antagonist. In another special aspect, the invention provides a method of preventing occurrence of and/or of treating cancer in a patient, said method comprising administration, either sequentially or simultaneously, of (i) a recombinant vector expressing in vivo at least one heterologous nucleotide sequence, especially an heterologous nucleotide sequence encoding at least one antigen and (ii) at least one glucocorticoid antagonist.

According to a preferred embodiment, said cancer is for example breast cancer, colon cancer, rectal cancer, lung cancer, cancer of the head and neck, renal cancer, malignant melanoma, laryngeal cancer, ovarian cancer, cervical cancer, prostate cancer.

In another aspect, the invention provides a method of preventing occurrence of and/or of treating infectious disease in a patient, said method comprising administration, either sequentially or simultaneously, of (i) an immunogenic composition and (ii) at least one glucocorticoid antagonist.

In one special aspect, the invention provides a method of '^reventin^¹ occurrence of and^or of treatin^g infectious disease in a patient, said method comprising administration, either sequentially or simultaneously, of (i) at least one antigen and (ii) at least one glucocorticoid antagonist.

In another special aspect, the invention provides a method of preventing occurrence of and/or of treating infectious disease in a patient, said method comprising administration, either sequentially or simultaneously, of (i) a recombinant vector expressing in vivo at least one heterologous nucleotide sequence, especially an heterologous nucleotide sequence encoding at least one antigen and (ii) at least one glucocorticoid antagonist. In special embodiments of the foregoing, the said antigen binds to MHC class I molecules (and is recognized by CD4+ T cells) .

In special embodiments of the foregoing, the said CD4+ T cell antigens are at least 10 amino acids, more preferably at least 13 amino acids in length, and can be much longer.

In special embodiments of the foregoing, the said immunogenic composition comprises a source of one or more CD4+ T cell epitopes of the target antigen (i.e. an epitope which binds to MHC class I molecules and is recognized by CD4+ T cells) .

According to a preferred embodiment, said infectious disease is a viral induced disease, such as for example disease induced by HIV, HCV, HBV, HPV, and the like.

In a further embodiment there is provided the use of a glucocorticoid antagonist as immunopotentiator, and more specifically as immuno-adjuvant . The term "immunopotentiator" is used to indicate any substance or preparation of substances which exhibits a high degree of potentiation, as one of its own function, in a humoral or cellular immune response system. An immunopotentiator which, when injected together with an antigen, increases the immune response to the antigen is generally called an "immuno-adjuvant".

According to preferred embodiment, there is provided the use of a glucocorticoid antagonist as immunopotentiator of CD4+ T cell response.

In a further embodiment there is provided the use of a glucocorticoid antagonist in the manufacture of an composition product for the enhancement of an immune response to an antigen, more specifically an antigen encoded by a recombinant vector, said antigen or recombinant vector being administered either sequentially or simultaneously with said antagonist. According to preferred embodiment, the said antigen binds to MHC class II molecules (and is recognized by CD4+ T cells) .

"Administered sequentially" means that the antigen and/or the recombinant vector [compound (i)] and the glucocorticoid antagonist [compound (ii) ] of the present composition product are administered independently from one another ; e.g. a first administration of one of the said compound and a separate second administration consisting in administration of the second compound. According to the present invention, the first administration can be done prior to, concurrently with or subsequent to the second administration, and vice-versa. The first and second administrations can be performed by different or identical delivery routes (systemic delivery and targeted delivery, or targeted deliveries for example). In one embodiment, each should be done into the same target tissue and most preferably by parenteral route. In a preferred embodiment, each should be delivered by independent routes and most preferably the glucocorticoid antagonist [compound (ii)] of the present composition product is administered by enteral route.

An "effective amount" or a "sufficient amount" of an active compound is an amount sufficient to effect beneficial or desired results, including clinical results. An effective amount can be administered in one or more administrations. A "therapeutically effective amount" is an amount to effect beneficial clinical results, including, but not limited to, alleviation of one or more symptoms associated with viral infection as well as prevention of disease (e.g. prevention of one or more symptoms of infection) . According to one preferred embodiment, "effective amount" or a "sufficient amount" of a glucocorticoid antagonist is an amount able to substantially decrease the blood level of corticosteroids, and more preferably the level of blood Cortisol, in a treated patient. "Substantially decrease" means that the blood glucocorticoid level after treatment with an effective amount of a glucocorticoid antagonist is below a baseline level determined in the zero-time blood sample (i.e. before the said treatment). For determining the blood Cortisol decrease observed with one said glucocorticoid antagonist, it is possible to take blood samples regularly (e.g. 3, 6, and 9 hours after administration of the compound) and to measure Cortisol level. According to the present invention, the glucocorticoid antagonists are able to obtain about 10% Cortisol level decreased , about 30%, preferably about 50%, more preferably at least 70% after 1, preferably 2 and more preferably 3 hours. Additionally, it is expected that repeated administration of glucocorticoid antagonists will lower the Cortisol level for longer periods of time. According to preferred embodiment, the blood level of corticosteroids, and more preferably the level of blood Cortisol, in a patient after treatment with the glucocorticoid antagonist of the invention is comprised between about 1 and about 5 μg/ml.

In preferred embodiment, the administration of the recombinant vector and/or antigen and of the glucocorticoid antagonist is substantially simultaneous.

In another embodiment, the glucocorticoid antagonist is administered before the administration of the recombinant vector and/or antigen. In this special embodiment, "before" means from about 5 min to about 2 weeks, more particularly from about 1 hour to about 1 week, more particularly from about 3 hours to about 48 hours.

The various components of the composition product of the invention (including antigen and/or recombinant vector and/or glucocorticoid antagonist) or its separate compounds (i) and (ii), are administered in patient as a pharmaceutically acceptable solution, which may routinely contain pharmaceutically-acceptable carrier (e.g. salt, buffering agents, preservatives, compatible carriers, adjuvants (e.g. alum, BCG, immune response modifiers) ) , and optionally other therapeutic ingredients.

The term pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the immunogenic combination also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical -efficiency.

The various components of the combination product of the invention (including antigen and/or recombinant vector and/or glucocorticoid antagonist), or its separate compounds (i) and (ii), can be administered by any ordinary route for administering medications. A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular immune composition, the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of an immune response without causing clinically unacceptable adverse effects. Preferred modes of administration are discussed herein. For use in therapy, an effective amount of the various components of the combination product of the invention (including antigen and/or recombinant vector and/or glucocorticoid antagonist) , or its separate compounds (i) and (ii) , can be administered to a subject by any mode that delivers the agent to the desired surface, e.g., mucosal, systemic and under any form, e.g. cream, solution.

The combination product, or its separate compounds (i) and (ii) , may be used according to the invention by a variety of modes of administration, including systemic, topical and localized administration. Injection can be performed by any means, for example by subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, intratumoral, intravascular, intraarterial injection (e.g. by hepatic artery infusion) or a vein feeding organ (e.g. injection into the portal vein) . Injections can be made with conventional syringes and needles, or any other appropriate devices available in the art. Alternatively the combination product (i.e. the immunogenic composition), or its separate compounds (i) and (ii) , may be administered via a mucosal route, such as the oral/alimentary, nasal, intratracheal, intrapulmonary, intravaginal or intra-rectal route. Topical administration can also be performed using transdermal means (e.g. patch, cream and the like) . In the context of the invention, intramuscular and subcutaneous administrations constitute the preferred routes for the immune composition, while the enteral route (esp. oral route) is preferred for the glucocorticoid antagonist .

For oral administration, the combination product of the invention (including antigen and/or recombinant vector and/or glucocorticoid antagonist) , or its separate compounds (i) and (ii) , can be formulated readily by combining the active compound (s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liguids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers. Recombinant viral vaccine which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

The combination product of the invention (including antigen and/or recombinant vector and/or glucocorticoid antagonist), or its separate compounds (i) and (ii) , when it is desirable to deliver it systemically, may be formulated for parenteral or enteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The combination product, or its separate compounds (i) and (ii) , may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Combination product, or its separate compounds (i) and (ii) , for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds (i) and/or (ii) may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds (i) and/or (ii) may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The recombinant viral vaccine may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the recombinant viral vaccine may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The recombinant viral vaccine also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Suitable liquid or solid combination product forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also ^"include granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.

The glucocorticoid antagonist may be administered per se or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v) ; citric acid and a salt (1-3% w/v) ; boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The combination product of the invention (including antigen . and/or recombinant vector and/or glucocorticoid antagonist), or its separate compounds (i) and (ii) , may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds (i) and (ii] into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Liquid dose units are vials or ampoules. Solid dose units are tablets, capsules and suppositories. For treatment of a patient, depending on activity of the compound, manner of administration, purpose of the immunization (i.e. prophylactic or therapeutic) , nature and severity of the disorder, age and body weight of the patient, different doses may be necessary. The administration of a given dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units. Multiple administrations of doses at specific intervals of weeks or months apart are usual for boosting the antigen- specific responses. Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compounds of the recombinant viral vaccine, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly (lactide-glycolide) , copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides . Microcapsules of the foregoing polymers containing drugs are described in, for example, US 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di-, and tri-glycerides; hydrogel release systems; sylastic systems; peptide bas_hed systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in US 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in US 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

The administration form of the recombinant vector and/or antigen [compound (i) ] and of the glucocorticoid antagonist [compound (ii)] can be identical or different for one said combination product according to the invention (e.g. compound (i) administered as a solution and compound (ii) administered as a tablet) . In other aspects, the invention relates to kits. One kit of the invention includes a container containing (i) at least one recombinant vector and/or antigen of the invention and a container containing (ii) at least one glucocorticoid antagonist and instructions for timing of administration of the compounds. The container may be a single container housing both (i) at least one recombinant vector and/or antigen of the invention and a container containing (ii) at least one glucocorticoid antagonist together or it may be multiple containers or chambers housing individual dosages of the compounds (i) and (ii), such as a blister pack. The kit also has instructions for timing of administration of the recombinant viral vaccine. The instructions would direct the subject to take the recombinant viral vaccine at the appropriate time. For instance, the appropriate time for delivery of the recombinant viral vaccine may be as the symptoms occur. Alternatively, the appropriate time for administration of the recombinant viral vaccine may be on a routine schedule such as monthly or yearly. The compounds (i) and (ii) may be administered simultaneously or separately as long as they are administered close enough in time to produce a synergistic immune response.

If desired, the method or use of the invention can be carried out in conjunction with one or more conventional therapeutic modalities (e.g. radiation, chemotherapy, hormonotherapy and/or surgery) . The use of multiple therapeutic approaches provides the patient with a broader based intervention. In one embodiment, the method of the invention can be preceded or followed by a surgical intervention. In another embodiment, it can be preceded or followed by radiotherapy (e.g. gamma radiation or electron beam therapy) . Those skilled in the art can readily formulate appropriate radiation therapy protocols and parameters which can be used (see for example Perez and Brady, 1992, Principles and Practice of Radiation Oncology, 2nd Ed. JB Lippincott Co; using appropriate adaptations and modifications as will be readily apparent to those skilled in the field) . In still another embodiment, the method or use of the invention is associated to chemotherapy (including anti-infectious therapy) with one or more drugs (e.g. drugs which are conventionally used for treating or preventing HPV infections, HPV-associated pathologic conditions).

The present Invention further concerns a method for improving the treatment of a cancer patient which is undergoing chemotherapeutic treatment with a chemotherapeutic agent, which comprises co-treatment of said patient along with a recombinant viral vaccine as above disclosed.

The present Invention further concerns a method of improving cytotoxic effectiveness of cytotoxic drugs or radiotherapy which comprises co-treating a patient in need of such treatment along with a combination product as above disclosed.

T r-> a n n f T -m -^ rrt- -t- H <_s m o -h Vi ^H Λ r n o --. r* -F -H Ηi __i i mron f i is carried out according to a prime boost therapeutic modality which comprises sequential administration of one or more primer composition (s) and one or more booster composition (s) . Typically, the priming and the boosting compositions use different vehicles which comprise or encode at least an antigenic domain in common. The priming composition is initially administered to the host organism and the boosting composition is subsequently administered to the same host organism after a period varying from one day to twelve months. The method of the invention may comprise one to about one hundred sequential administrations of the priming composition followed by one to about one hundred sequential administrations of the boosting composition; preferably one to about fifty sequential administrations of the priming composition followed by one to about fifty sequential administrations of the boosting composition; even more preferably one to about ten sequential administrations of the priming composition followed by one to about ten sequential administrations of the boosting composition. Desirably, injection intervals are a matter of one week to six months. Moreover, the priming and boosting compositions can be administered at the same site or at alternative sites by the same route or by different routes of administration. For example, compositions based on HPV early polypeptide can be administered by a mucosal route whereas recombinant viral vaccine is preferably injected, e.g. subcutaneous injection for a MVA vector.

The ability to induce or stimulate an immune response (e.g. anti-HPV or anti-HCV immune response) upon administration in a patient can be evaluated either in vitro or in vivo using a variety of assays which are standard in the art. For a general description of techniques available to evaluate the onset and activation of an immune response, see for example Coligan et al. (1992 and 1994, Current Protocols in Immunology ; ed J Wiley & Sons Inc, National Institute of Health) . Measurement of cellular immunity can be performed by measurement of cytokine profiles secreted by activated effector cells including those derived from CD4+ and CD8+ T- cells (e.g. quantification of IL-IO or IFN gamma-producing cells by ELIspot), by determination of the activation status of immune effector cells (e.g. T cell proliferation assays by a classical [³H] thymidine uptake) , by assaying for antigen- specific T lymphocytes in a sensitized subject (e.g. peptide- specific lysis in a cytotoxicity assay) . The ability to stimulate a humoral response may be determined by antibody binding and/or competition in binding (see for example Harlow, 1989, Antibodies, Cold Spring Harbor Press) . The method of the invention can also be further validated in animal models challenged with an appropriate tumor-inducing agent (e.g. HPV- E6 and E7-expressing TCl cells) to determine anti-tumor activity, reflecting an induction or an enhancement of an anti-HPV immune response.

Disease conditions which may especially be treated in accordance with the present invention are for example cervical cancer or precursor lesions of this malignant neoplasia, which are called cervical intraepithelial neoplasia (CIN) or squamous intraepithelial lesions (SIL) . The immunogenic combination of the invention may also be useful in the treatment of asymptomatic infections of the cervix in patients identified by DNA diagnosis, or asymptomatic infections that are assumed to remain after surgical treatment of cervical cancer, CIN or SIL, or asymptomatic infections presumed to exist following epidemiological reasoning. The disease conditions to be treated also include genital warts, and common warts and plantar warts. All of these conditions are also caused by a large number of other HPV types, and the agents, compounds and methods of the invention may also be usefully directed against these viruses. All of these lesions presumably derive from asymptomatic infections, that are most often not diagnosed. The present invention may also be usefully targeted against all of these asymptomatic infections.

The combination product of the invention, or 'its separate compounds (i) and (ii) , may be employed in methods for treating a variety of diseases and pathologic conditions, especially those associated with an HCV infection. It is especially useful for treating HCV persistent infection and liver cancer in HCV-infected patients. The term "cancer" encompasses any cancerous conditions including diffuse or localized tumors, metastasis, cancerous polyps as well as preneoplastic lesions (e.g. cirrhosis). Preferably, upon introduction into a host organism according to the modalities described herein, the combination product of the invention, or its separate compounds (i) and (ii) , provides a therapeutic benefit to the treated host. The therapeutic benefit can be evidenced by a number of ways, for instance a decrease of HCV viremia detected in blood, plasma or sera of an infected individual as compared to before treatment, and/or by the detection of an anti-HCV immune response (e.g. production of anti-HCV antibodies and/or T cell-mediated immunity) or by the delay of the symptoms associated with an HCV infection (e.g. delay in the development of liver cirrhosis or cancer) , or by a decrease of liver inflammation/steatosis/fibrosis conditions typically associated with HCV infection or by an improved response of the individual to conventional therapies.

In still another embodiment, the method or use of the invention is associated to chemotherapy with one or more HCV drugs which are conventionally used for treating or preventing HCV infections, HCV-associated diseases and pathologic conditions. Representative examples of HCV drugs include without limitation protease inhibitors (e.g. serine protease inhibitors such as VX950 of Vertex) , polymerase inhibitors, helicase inhibitors, antifibrotics, nucleoside analogs, TLR agonists, N-glycosylation inhibitors, siRNA, antisense oligonucleotides, anti-HCV antibodies, immune modulators, therapeutic vaccines and antitumor agents usually used in the treatment of HCV-associated hepatocarcinomas (e.g. adriamycin or a mixture of adriamycin lipiodol and spongel usually administered by chimioembolisation in the hepatic artery) . For example, therapeutic vaccines include recombinant antigens, VLPs, vectors or synthetic peptides based on or encoding HCV structural proteins (Core, envelope El and/or E2 ) which are particularly suited to trigger an anti-HCV humoral response. Such HCV drugs can be provided in a single dose or, alternatively, in multiple doses according to standard protocols, dosages and regimens over several hours, days and/or weeks. Their administration may precede, be concomitant, or subsequent to the administration of the combination product of the invention, or its separate compounds (i) and (ii) . A particularly suitable combination includes treatment with pegylated IFN-α2a (e.g. at a dose of lOμg/week) and/or ribavirin (e.g. at 800 to 1200 mg/day) for 24 to 48 weeks, before, in parallel or subsequently to the method of the invention.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced in a different way from what is specifically described herein.

All of the above cited disclosures of patents, publications and database entries are specifically incorporated herein by reference in their entirety to the same extent as if each such individual patent, publication or entry were specifically and individually indicated to be incorporated by reference.

EXAMPLES

Adjuvant effect of glucocorticoid antagonists according to the Invention on immunotherapy of cancer. Glucocorticoids are known as immunosuppressive drugs. Natural glucocorticoids may have a role in controlling the immune response. Inhibiting this effect could in turn enhance the immune response against certain antigens such as tumor antigens, viral or bacterial antigens. To test this concept, we have used two glucocorticoid inhibitors in a model of cancer immunotherapy.

Metyrapone is an inhibitor of endogenous adrenal corticosteroid synthesis (figure 1). Metyrapone is a drug used in the diagnosis of adrenal insufficiency and occasionally in the treatment of hypercorticolism. It blocks Cortisol synthesis by inhibiting steroid 11-beta-hydroxylase. This stimulates ACTH secretion, which in turn increases plasma 11- deoxycortisol levels. When excessive ACTH secretion is the cause of hypercortisolism, the metyrapone test helps clarify if the source of the ACTH- is pituitary or not.

Mifepristone is a synthetic steroid compound used as a pharmaceutical (figure 2). It is used as an abortive agent in the first two months of pregnancy, and in smaller doses as an emergency contraceptive. It can also be used as a treatment for obstetric bleeding. In the presence of progesterone, mifepristone acts as a competitive receptor antagonist at the progesterone receptor while in the absence of progesterone, lifepristone acts as a partial agonist. In addition to being an antiprogesterogen, mifepristone is also an antiglucocorticoid

To test the invention, six groups of 13 six week old female B6D2 mice received subcutaneoulsy 3 x 10⁵ RenCa cells stably expressing the human MUCl molecule. Three groups received nothing while the 3 other groups received MVATG9931 (MVA expressing human MUCl) on days 4, 11 and 18 (5xlO⁷ PFU/100 μL) . Each of the three groups remained either untreated or received metyrapone (50mg/Kg) or mifepristone (40 Mg/Kg) on days 2-4, 7-11, 14-18 and 21-25 by intraperitoneal injection. Animals survival was monitered (figure 3) .

Survival was significantly improved in the group receiving Metyrapone and the MVA vaccine relative to the naive control group (p<0.003) at the end of the experiment while no statistically significant difference was seen between the other groups

We also looked at the MUCl-specific immune response as measured by the frequency of Interferon-gamma (IFNgamma) producing cells/10⁶ splenocytes in an ELISPOT assay. To this end, spleens were taken from non-tumor bearing animals on the last day of anti-corticoid treatment and incubated with either MHC class I immunodominant MUCl peptides (S9L2, L9V) or MHC class II immunodominant MUCl peptides (T24P, G23D) . As seen in figure 4, the combination of Mifepristone with a MVA vector expressing the MUCl protein biases the population on IFNgamma- producing cells to a class II restricted population.

Claims

1. Combination product containing (i) at least one antigen and (ii) at least one glucocorticoid antagonist.

2. A combination product containing (i) at least one recombinant vector expressing in vivo at least one heterologous nucleotide sequence encoding an antigen of claim 1, and (ii) at least one glucocorticoid antagonist.

3. A combination product of claim 1 or 2, wherein said antigen binds to MHC class II molecules and is recognized by CD4+ T cells.

4. A combination product of claim 3, wherein it is at least 10 amino acids in length.

5. A combination product of claim 3, wherein it is at least 13 amino acids in length. β. Use of glucocorticoid antagonists for the preparation of combination product of claim 1 to 3, wherein said glucocorticoid antagonists elicit an enhanced immune response to at least one antigen administered to a patient.

7. The use of claim 6, wherein the said enhanced immune response is a CD4+ T cell response.

8. Use of a glucocorticoid antagonist as immunopotentiator .

9. The use of claim 8 wherein said glucocorticoid antagonist is an immunopotentiator of CD4+ T cell response.

10. A combination product of any claim 1 to 5, wherein the glucocorticoid antagonist is a compound or agent able to reduce, and preferably to inhibit, the ACTH release by the hypophysis.

11. A combination product of claim 10, wherein the glucocorticoid antagonist is selected in the group consisting of a corticotropin-releasing factor (CRF) receptorantagonist, cyproheptadine, desmethylcyproheptadine , bromocriptine , valproic acid and derivatives, analogues, solvates or salts thereof.

12. A combination product of any claim 1 to 5, wherein the glucocorticoid antagonist is a compound or agent able to reduce corticosteroids synthesis.

13. A combination product of claim 12, wherein the glucocorticoid antagonist is selected in the group consisting of 1- (o-chlorophenyl) -1- (p-chlorophenyl) -2, 2- dichloroethane (o,p'-DDD) ; 1- (o-chlorophenyl) -1- (p- chlorophenyl) -2, 2-dichloropropane; aminogluthetimide; ketoconazole and its derivatives ; econazole; miconazole; metyrapone ; trilostane and its derivatives (2 alpha- cyano-4alpha, 5alpha-epoxyandrostan-17beta-ol-3-one; derivatives of diphenylmethane; derivatives of pyridine ; substituted alpha alpha glutaramide; steroid of the spironolactone family; synthetic steroid ZK91587 ; etomidate ; and derivatives, analogues, solvates or salts thereof.

14. A combination product of any claim 1 to 5, wherein the glucocorticoid antagonist is a cortisol-sequestering agent .

15. A combination product of claim 14, wherein the glucocorticoid antagonist is selected in the group consisting of cyclodextrins and derivatives, analogues, solvates or salts thereof.

16. A combination product of any claim 1 to 5, wherein the glucocorticoid antagonist is a compound or agent which limits the biological effects of glucocorticoid by inhibiting glucocorticoid receptors.

17. A combination product of claim 16, wherein the glucocorticoid antagonist is selected in the group consisting of 17 β-hydroxy-llβ- (4-dimethylaminophenyl) - 17α- (1-propynyl) estra-4 , 9-dien-3-one; non steroid substance or acetylsalicic acid ; 21-hydroxy 6,19 oxidoprogesterone; and derivatives, analogues, solvates or salts thereof.

18. A combination product of any claim 1 to 5, wherein the glucocorticoid antagonist is a compound or agent selected in the group consisting in enkephalin analogue [D-Ala2, MePhe4, Met(o)-ol] enkephalin (DAMME) ; loperamide; dexamethasone ; alprazolam; clonidine ; oxytocin; 6- Mercaptonicotinic and derivatives, analogues, solvates or salts thereof.